Methods for treating cancer using 6,8-bis (benzylthio) octanonic acid

ABSTRACT

Pharmaceutical formulations containing lipoic acid derivatives and ion pairs thereof are described. The pharmaceutical formulations are useful in the treatment of medical disorders, such as cancer.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/662,612, filed Jul. 28, 2017, which is a continuation of U.S. patentapplication Ser. No. 14/857,192, filed Sep. 17, 2015, which is acontinuation of U.S. patent application Ser. No. 13/569,654, filed Aug.8, 2012, which is a continuation of U.S. patent application Ser. No.12/604,763, filed Oct. 23, 2009, now U.S. Pat. No. 8,263,653, which is acontinuation-in-part of U.S. patent application Ser. No. 12/105,100,filed, Apr. 17, 2008, which claims the benefit of and priority to U.S.Provisional Patent Application Ser. No. 60/912,605, filed Apr. 18, 2007;the entire contents of each of which are hereby incorporated byreference for all purposes.

FIELD OF THE INVENTION

The present invention is directed to pharmaceutical formulationscontaining lipoic acid derivatives or salts thereof which selectivelykill tumor cells by altering cancer cell metabolism and signaltransduction pathways linked to the Warburg Effect, as well as tomethods of treating a subject with such pharmaceutical formulations.

BACKGROUND

All mammalian cells require energy to live and grow. Cells obtain thisenergy by metabolizing food molecules by oxidative metabolism. The vastmajority of normal cells utilize a single metabolic pathway tometabolize their food. The first step in this metabolic pathway is thepartial degradation of glucose molecules to pyruvate in a process knownas glycolysis which yields two ATP units. Glycolysis can occur evenunder hypoxic conditions. Pyruvate is further degraded in themitochondrion by a process known as the tricarboxylic acid (TCA) cycleto produce thirty-six ATP units per glucose molecule, water and carbondioxide. The TCA cycle requires oxygen. During periods of reduced oxygenlevels, normal cells adapt by a variety of mechanisms and return tonormal metabolism as oxygen levels are restored. A critical link betweenglycolysis and the TCA cycle is an enzyme known as pyruvatedehydrogenase (“PDH”). PDH is part of a larger multi-subunit complex(hereinafter “PDC”). PDH, in conjunction with other enzymes of the PDCcomplex, produces acetyl CoA which effectively funnelsglycolysis-produced pyruvate to the TCA cycle.

Most cancers display profound perturbation of energy metabolism. One ofthe fundamental changes is the adoption of the Warburg Effect, whereglycolysis becomes the main source of ATP. An ATP deficit followsreduced TCA ATP generation. In other words, cancer cells behave as ifthey are hypoxic even when they are not. This change in energymetabolism represents one of the most robust and well-documentedcorrelates of malignant transformation and has been linked to otherchanges resulting in tumor growth and metastasis. Because of the reducedlevels of ATP available as a result of glycolysis largely beingde-linked from the TCA cycle, cancer cells increase their uptake ofglucose and its conversion to pyruvate in an attempt to make up theenergy deficit. Excess pyruvate and other metabolic by-products of theWarburg biochemistry must be managed. A number of these metabolites areknown to be cytotoxic, e.g., acetaldehyde. PDC in cancer along withother related enzymes plays a major role in managing and/or detoxifyingthe excess pyruvate and metabolites. For example, the joining of twoacetyl molecules to form the neutral compound acetoin. This generationof acetoin is catalyzed by a tumor-specific form of PDC. It has beensuggested that lipoic acid acts as a cofactor with PDC and relatedlipoamide using enzymes in detoxifying these otherwise toxicmetabolites. Whether lipoic acid is made by healthy and cancer cells orwhether it is an essential nutrient is debated in the literature, andboth may be the case. The genes required to produce lipoic acid havebeen identified in mammalian cells. Whether mitochondrial pumps oruptake mechanisms are present in healthy or cancer cells or whether theydiffer in diverse tissues is not known. Although the TCA cycle stillfunctions in cancer cells, the tumor cell TCA cycle is a variant cyclewhich depends on glutamine as the primary energy source. Inhibition orinactivation of tumor-specific PDC and related enzymes that detoxifymetabolites may promote apoptosis or necrosis and cell death.

Despite extensive work characterizing the highly conserved changes amongdiverse tumor types and their metabolism, the changes remain to besuccessfully exploited as a target for cancer chemotherapy. As cancerremains the number two killer of Americans, there is an urgent need fornew approaches to disease management. It has been suggested that lipoicacid due to its redox potential properties may be useful in thetreatment of diverse diseases involving mitochondrial function such asdiabetes, Alzheimers disease and cancer. These reports teach that theavailability of the redox shift from SH to S—S be maintained to have thedesired effect.

U.S. Pat. Nos. 6,331,559 and 6,951,887 disclose a novel class oftherapeutic agents which selectively targets and kills tumor cells andcertain other types of diseased cells. These patents further disclosepharmaceutical compositions comprising an effective amount of a lipoicacid derivative according to its invention along with a pharmaceuticallyacceptable carrier. However, these patents provide no specific guidancewith regard to the selection of suitable pharmaceutically acceptablecarriers. As the present inventors have now discovered, thepharmaceutical formulation of the lipoic acid derivatives has provedpivotal in achieving efficacy for these agents.

SUMMARY

The invention generally provides pharmaceutical formulations suited fordelivering a lipoic acid derivative or salt thereof to a subject in needof treatment, such as in need of cancer treatment. The pharmaceuticalformulations comprise, in certain embodiments, a pharmaceuticallyacceptable carrier and an ion pair formed by a lipoic acid derivativeand ion pairing agent. The pharmaceutical formulations comprising theion pair can help improve the solubility and/or efficacy of the lipoicacid derivative for use in treating, e.g., cancer. Accordingly, oneaspect of the invention provides a pharmaceutical formulationcomprising: (a) a pharmaceutically acceptable diluent; and (b) an ionpair formed by a lipoic acid derivative and an ion pairing agent;wherein the lipoic acid derivative is a compound of Formula Irepresented by

wherein the variables are as defined in the detailed description herein.In another aspect, the invention provides a pharmaceutical formulationcomprising bis-benzyl lipoate, and triethanolamine. In another aspect,the invention provides an intravenous pharmaceutical composition fortreating cancer, wherein the composition comprises6,8-bis(benzylthio)octanoic acid or an ion pair thereof in an amounteffective for treating cancer, and a pharmaceutically acceptable aqueousdiluent for solubilizing 6,8-bis(benzylthio)octanoic acid or an ion pairthereof.

In another aspect, the invention provides a method of treating orpreventing a disease characterized by disease cells that are sensitiveto lipoic acid derivatives. The method comprises administering to apatient in need thereof a pharmaceutical formulation described herein totreat or prevent said disease. In certain embodiments, the disease isselected from the group consisting of carcinoma, sarcoma, myeloma,lymphoma, leukemia and mixed types thereof.

In another aspect, the invention provides a method of inducing necroticdeath of a cancer cell. The method comprises administering to a cancercell an effective amount of a pharmaceutical formulation comprising anion pair formed by a lipoic acid derivative and an organic Bronsted basecompound. In another aspect, the invention provides a method of inducingapoptotic death of a cancer cell. The method comprises administering toa cancer cell an effective amount of a pharmaceutical formulationcomprising an ion pair formed by a lipoic acid derivative describedherein and an ion pairing agent that is an alkali metal hydroxide or analkaline earth metal hydroxide.

Yet another aspect of the invention provides an ion pair consisting of:at least one ion pairing agent selected from the group consisting oftriethanolamine, polyethyleneimine, diethanolamine, monoethanolamine,mefenamic acid, tromethamine, ethanolamine, diethanolamine,ethylenediamine, lysine, and diethylamine; and at least one lipoic acidderivative represented by Formula (I):

wherein the variables are as described in the detailed description.

In another aspect, the present invention is directed to a pharmaceuticalformulation comprising (a) at least one lipoic acid derivative or saltthereof and (b) at least one ion pairing agent and optionally (c) apharmaceutically acceptable diluent. In a preferred embodiment of theinvention, the lipoic acid derivative has the formula (I):

wherein R₁ and R₂ are independently selected from the group consistingof acyl defined as R₃C(O)—, alkyl defined as C_(n)H_(2n+1), alkenyldefined as C_(m)H_(2m−1), alkynyl defined as C_(m)H_(2m−3), aryl,heteroaryl, alkyl sulfide defined as CH₃(CH₂)_(n)—S—, imidoyl defined asR₃C(═NH)—, hemiacetal defined as R₄CH(OH)—S—, and hydrogen provided thatat least one of R₁ and R₂ is not hydrogen; wherein R₁ and R₂ as definedabove can be unsubstituted or substituted; wherein R₃ is hydrogen,alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylaryl, heteroaryl, orheterocyclyl, any of which can be substituted or unsubstituted; whereinR₄ is CCl₃ or COOH; and wherein x is 0-16, n is 0-10 and m is 2-10. In apreferred embodiment, R₁ and R₂ are both a benzyl group, i.e., both R₁and R₂ are independently —CH₂C₆H₅. In another preferred embodiment, thelipoic acid derivative has the formula (II):

wherein M is a metal chelate, —[C(R₁)(R₂)]_(z)— or other metal complex;wherein R₁ and R₂ are independently selected from the group consistingof acyl defined as R₃C(O)—, alkyl defined as C_(n)H_(2n+1), alkenyldefined as C_(m)H_(2m−1), alkynyl defined as C_(m)H_(2m−3), aryl, alkylsulfide defined as CH₃(CH₂)_(n)—S—, imidoyl defined as R₃C(═NH)—,hemiacetal defined as R₄CH(OH)—S— and hydrogen; wherein R₁ and R₂ asdefined above can be unsubstituted or substituted; wherein R₃ ishydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylaryl,heteroaryl, or heterocyclyl, any of which can be substituted orunsubstituted; wherein R₄ is CCl₃ or COOH; and wherein x is 0-16, z is0-5, n is 0-10 and m is 2-10.

Further preferred embodiments of this invention include those in whichthe lipoic acid derivative is present in a therapeutically effectiveamount. Still further preferred embodiments of this invention includethose in which the ion pairing agent is selected from the groupconsisting of triethanolamine, polyethyleneimine, monoethanolamine,diethanolamine, mefanamic acid, tromethamine and combinations thereof,those in which the ion pairing agent is a polymer-conjugated ion pairingagent, and those in which the ion pairing agent and the at least onelipoic acid derivative is present in a ratio ranging from about 1000:1to about 1:1000. Further preferred embodiments of the present inventionalso include those in which the diluent is selected from the groupconsisting of saline, a sugar solution, an alcohol, dimethylformamide,dimethylsulfoxide, dimethylacetamide and combinations thereof.

In another aspect, the present invention is directed to a method oftreating a disease characterized by disease cells that are sensitive tolipoic acid derivatives comprising administering to a patient in needthereof a pharmaceutical formulation comprising at least one lipoic acidderivative or salt thereof, at least one ion pairing agent, andoptionally a pharmaceutically acceptable diluent. In yet another aspect,the present invention is directed to a method of preventing a diseasecharacterized by disease cells that are sensitive to lipoic acidderivatives comprising administering to a patient in need thereof apharmaceutical formulation comprising at least one lipoic acidderivative, at least one ion pairing agent, and optionally apharmaceutically acceptable diluent. In preferred embodiments of thesemethods, the disease is a cancer such as a carcinoma, sarcoma, myeloma,lymphoma, leukemia, or a mixed cancer type.

In yet another aspect, the invention is directed to an ion pairconsisting of (a) at least one lipoic acid derivative and (b) at leastone ion pairing agent, most preferably bis-benzyl lipoate andtriethanolamine, respectively.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the tumor volume and body weight, respectively, inH-460 tumor-bearing mice treated with bis-benzyl lipoate in a Tween80/ethanol pharmaceutical formulation.

FIGS. 2A, 2B and 2C show the tumor volume in H-460 tumor-bearing micetreated with bis-benzyl lipoate in a triethanolamine/dextrosepharmaceutical formulation at 3 different dosage levels.

FIG. 3 shows percent viability of A2780 and BXPC3 tumor cells treatedwith either CPI-613 in triethanolamine or CPI-613 in ethanolamine.

FIG. 4 shows percent viability of A2780 and BXPC3 tumor cells treatedwith either CPI-613 in triethanolamine or CPI-613 in diethanolamine.

FIG. 5 shows percent viability of A2780 and BXPC3 tumor cells treatedwith either CPI-613 in triethanolamine or CPI-613 in meglumine.

FIG. 6 shows percent viability of A2780 and BXPC3 tumor cells treatedwith either CPI-613 in triethanolamine or CPI-613 in 3-amino-1-propanol.

FIG. 7 shows percent viability of A2780 and BXPC3 tumor cells treatedwith either CPI-613 in triethanolamine or CPI-613 in triethylamine.

FIG. 8 shows percent viability of A2780 and BXPC3 tumor cells treatedwith either CPI-613 in triethanolamine or CPI-613 in ethylenediamine.

FIG. 9 shows percent viability of A2780 and BXPC3 tumor cells treatedwith either CPI-613 in triethanolamine or CPI-613 in lysine.

FIG. 10 shows percent viability of A2780 and BXPC3 tumor cells treatedwith either CPI-613 in triethanolamine or CPI-613 in diisopropanolamine.

FIG. 11 shows percent viability of A2780 and BXPC3 tumor cells treatedwith either CPI-613 in triethanolamine or CPI-613 in diethylamine.

FIG. 12 shows percent viability of A2780 and BXPC3 tumor cells treatedwith either CPI-613 in triethanolamine or CPI-613 sodium salt.

FIG. 13 shows percent viability of A2780 and BXPC3 tumor cells treatedwith either CPI-613 in triethanolamine or CPI-613 potassium salt.

FIG. 14 shows percent viability of A2780 and BXPC3 tumor cells treatedwith either CPI-613 in triethanolamine or CPI-613 in ammonium hydroxide.

FIG. 15 is a bar graph showing the percentage of dead cells and themechanism of cell death (i.e., apoptosis or necrosis) followingadministration of CPI-613 in triethanolamine or CPI-613 sodium salt.

FIG. 16 is a bar graph showing the percentage of dead cells and themechanism of cell death (i.e., apoptosis or necrosis) followingadministration of CPI-613 in triethanolamine or CPI-613 sodium salt.

FIG. 17 is a bar graph showing the percentage of live cells followingadministration of CPI-613 in triethanolamine or CPI-613 sodium salt.

DETAILED DESCRIPTION

The present invention is directed to pharmaceutical formulationscontaining lipoic acid derivatives which are effective to target andkill tumor cells. While the pharmaceutical formulation of manytherapeutic agents is quite conventional, the present inventors havefound that the pharmaceutical formulation of lipoic acid derivatives isnot. In fact, the particular pharmaceutical formulation in which alipoic acid derivative is placed may well be the determining factorbetween inactivity and activity for its intended purpose. Accordingly,one aspect of the invention provides a pharmaceutical formulationcomprising (a) at least one lipoic acid derivative and (b) at least oneion pairing agent and optionally (c) a pharmaceutically acceptablediluent, as described in detail herein.

I. Lipoic Acid Derivatives and Ion Pairs Thereof

Lipoic acid derivatives suitable for use in the present inventioninclude those described in full detail in each of U.S. Pat. Nos.6,331,559 and 6,951,887 and those described in U.S. ProvisionalApplication No. 60/912,598, filed Apr. 18, 2007 and correspondingco-pending U.S. patent application Ser. No. 12/105,096, filed Apr. 18,2008, the disclosure of each of which is incorporated by referenceherein. Lipoic acid derivatives suitable for use in the presentinvention can be made according to known procedures such as those setforth in the aforementioned patents. In a preferred embodiment of thisinvention, the lipoic acid derivative has the formula (I):

or a salt thereof;

wherein R₁ and R₂ are independently selected from the group consistingof acyl defined as R₃C(O), alkyl defined as C_(n)H_(2n+1), alkenyldefined as C_(m)H_(2m−1), alkynyl defined as C_(m)H_(2m−3), aryl,heteroaryl, alkyl sulfide defined as CH₃(CH₂)_(n)—S—, imidoyl defined asR₃C(═NH)—, hemiacetal defined as R₄CH(OH)—S—, and hydrogen provided thatat least one of R₁ and R₂ is not hydrogen;

wherein R₁ and R₂ as defined above can be unsubstituted or substituted;

wherein R₃ is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl,alkylaryl, heteroaryl, or heterocyclyl, any of which can be substitutedor unsubstituted;

wherein R₄ is CCl₃ or COOH; and

wherein x is 0-16, n is 0-10 and m is 2-10.

As used herein, acyl refers to an R₃C(O)— group, where R₃ can be,without limitation, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl,alkylaryl, heteroaryl, or heterocyclyl, any of which can be substitutedor unsubstituted. In other words, one of the listed R₃ groups is linkedto the carbon backbone of formula (I) through a thio-ester linkage.Examples of acyl groups include, without limitation, acetyl, benzoyl andbenzoyl derivatives, 4-fluorobenzoyl and 1-methylpyrrole-2-carboxyl.Specific examples of lipoic acid derivatives containing an acyl groupinclude, without limitation, bis-acetyl lipoate and bis-benzoyl lipoate.

As used herein, alkyl refers to a C_(n)H_(2n+1) group, wherein n is1-10, more preferably 1-6 and most preferably 1-4, i.e., an alkyl grouplinked to the carbon backbone of formula (I) through a thio-etherlinkage. Alkyl groups can be either aliphatic (straight or branchedchain) or alicyclic; alicyclic groups may have additions orsubstitutions on any of the carbons to form heterocyclics. At least oneheteroatom such as N, O or S may be present in a given alkyl group,i.e., in the carbon chain. Alkyl groups may be substituted orunsubstituted on any of their carbons. A preferred alkyl group is analkyl group substituted with an aryl or heteroaryl group, i.e., whereinR₁ or R₂ is an alkylaryl or alkylheteroaryl group; the aryl orheteroaryl group may be substituted or unsubstituted. Examples of alkylgroups include, without limitation, methyl, ethyl, butyl, decanyl,cyclopropyl, 4-pyridine methyl, 2-anthraquinone methyl,N-phenylacetamide, phenylethyl, 2-ethanoic acid, 2-acetamido,4-(2-acetamido-pyridinyl)methyl, N-[(2-fluorophenyl)methyl]acetamide,N-[(6-methoxy-3-pyridyl)methyl]acetamide,5-(acetylamino)pyridine-2-carboxamide,5-(6,8-diaza-7-oxo-3-thiabicyclo[3.3.0]oct-2-yl)-N-(2-carbonylaminoethyl)pentanamideand 5-(6,8-diaza-7-oxo-3-thiabicyclo[3.3.0]oct-2-yl)pentacarboxyl.Specific examples of lipoic acid derivatives containing an alkyl groupinclude, without limitation, 6,8-bis carbamoyl methylipoate and 6,8methyl-succinimido lipoate.

As used herein, alkenyl refers to a C_(m)H_(2m−1) group, wherein m is2-10, i.e., an alkenyl group linked to the carbon backbone of formula(I) through a thio-ether linkage. Alkenyl groups can be either aliphatic(straight or branched chain) or alicyclic; alicyclic groups may haveadditions or substitutions on any of the carbons to form heterocyclics.At least one heteroatom such as N, O or S may be present in a givenalkenyl group, i.e., in the carbon chain. Alkenyl groups may besubstituted or unsubstituted on any of their carbons. Examples ofalkenyl groups include, without limitation, propenyl, 2,3dimethyl-2-butenyl, heptenyl and cyclopentenyl.

As used herein, alkynyl refers to a C_(m)H_(2m−3), where m is 2-10,i.e., an alkynyl group linked to the carbon backbone of formula (I)through a thio-ether linkage. Alkynyl groups can be either aliphatic(straight or branched chain) or alicyclic; alicyclic groups may haveadditions or substitutions on any of the carbons to form heterocyclics.At least one heteroatom such as N, O or S may be present in a givenalkynyl group, i.e., in the carbon chain. Alkynyl groups may besubstituted or unsubstituted on any of their carbons. Examples ofalkynyl groups include, without limitation, acetylenyl, propynyl andoctynyl.

As used herein, aryl refers to an aromatic or aryl group linked to thecarbon backbone of formula (I) through a thio-ether linkage. Aryl ispreferably an unsaturated ring system having 6-10 carbon atoms. Arylalso includes organometallic aryl groups such as ferrocene. Aryl groupsmay be substituted or unsubstituted on any of their carbons. Examples ofaryl groups include, without limitation, benzyl (—CH₂C₆H₅), benzylderivatives such as methylbenzyl and aminobenzyl,(1,2,3,4,5-pentafluorophenyl)methyl, triphenylmethyl, 4-methy benzoicacid, ferrocene methyl, 2-naphthylmethyl, 4,4-biphenylmethyl, andstilbene (or 1-((1E)-2-phenylvinyl)-4-methyl benzene). A specificexample of a lipoic acid derivative containing an aryl group isbis-benzyl lipoate.

As used herein, heteroaryl refers to an aromatic heterocyclic ringsystem (monocyclic or bicyclic) where the heteroaryl moieties are fiveor six membered rings containing 1 to 4 heteroatoms selected from thegroup consisting of S, N, and O; the heteroaryl group is linked to thecarbon backbone of formula (I) through a thio-ether linkage. Heteroarylgroups may be substituted or unsubstituted on any of their atomsespecially on the carbon atoms. Examples of heteroaryl groups include,without limitation, benzothiazole, quinoline, 7-chloroquinoline, furan,thiophene, indole, azaindole, oxazole, thiazole, isoxazole, isothiazole,imidazole, N-methylimidazole, pyridine, pyrimidine, pyrazine, pyrrole,N-methylpyrrole, pyrazole, N-methylpyrazole, 1,3,4-oxadiazole,1,2,4-triazole, 1-methyl-1,2,4-triazole, 1H-tetrazole,1-methyltetrazole, benzoxazole, benzofuran, benzisoxazole,benzimidazole, N-methylbenzimidazole, azabenzimidazole, indazole,quinazoline and pyrrolidinyl.

As used herein, alkyl sulfide refers to a CH₃(CH₂)_(n)—S— group, where nis 0-9. In other words, an alkyl group is linked to the carbon backboneof formula (I) through a disulfide linkage. The alkyl group (i.e.,CH₃(CH₂)_(n)) can be substituted or unsubstituted on any of its carbonsand shares the same features as set forth above with regard to theC_(n)H_(2n+1) alkyl group.

As used herein, imidoyl refers to a R₃C(═NH)— group, where R₃ can be,without limitation, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl,alkylaryl, heteroaryl, or heterocyclyl, any of which can be substitutedor unsubstituted. In other words, one of the listed R₃ groups is linkedto the carbon backbone of formula (I) through a thio-imide linkage.

As used herein, hemiacetal refers to an R₄CH(OH)—S— group, where R₄ is acompound with strongly electron withdrawing substituents such as,without limitation, CF₃, CCl₃ or COOH.

Any of the above-described groups can be unsubstituted or substituted.Exemplary substituents include, without limitation, alkyl, alkenyl,alkynyl, aryl, heteroaryl, acyl, alkoxycarbonyl, alkoxy, alkoxyalkyl,alkoxyalkoxy, cyano, halogen, hydroxy, nitro, oxo, trifluoromethyl,trifluoromethoxy, trifluoropropyl, amino, amido, alkylamino,dialkylamino, dialkylaminoalkyl, hydroxyalkyl, alkoxyalkyl, alkylthio,—SO₃H, —SO₂NH₂, —SO₂NHalkyl, —SO₂N(alkyl)₂, —CO₂H, CO₂NH₂, CO₂NHalkyl,and —CO₂N(alkyl)₂. In addition, any number of substitutions may be madeon any of the above-described groups; in other words, it is possible tohave a mono-, di-, tri-, etc. substituted R₁ or R₂ group, and thesubstituents themselves may also be substituted. Further, any of the R₁or R₂ groups may be appropriately generally substituted with any of acarbohydrate, a lipid, a nucleic acid, an amino acid or a polymer of anyof those, or a single or branched chain synthetic polymer (having amolecular weight ranging from about 350 to about 40,000).

For any definition of R₁ and R₂ noted above, the thio-ester orthio-ether linkage by which the R₁ and R₂ are linked to the backbone canbe oxidized to produce sulfoxides or sulfones; in other words, the —S—in the linkage could be —S(O)— or —S(O)₂. In addition, for anydefinition of R₁ and R₂ noted above, the thio-ester or thio-etherlinkage by which the R₁ and R₂ are linked to the backbone may furthercomprise disulfides that can be oxidized to thiosulfinic or thiosulfonicacids; in other words, instead of —S— in a linkage, the linkage could be—S(O)—S— or —S(O)₂—S—.

In another preferred embodiment of this invention, the lipoic acidderivative has the formula (II):

M is a metal chelate, —[C(R₁)(R₂)]_(z)— or other metal complex. R₁ andR₂ are independently selected from the group consisting of acyl definedas R₃C(O)—, alkyl defined as C_(n)H_(2n+1), alkenyl defined asC_(m)H_(2m−1), alkynyl defined as C_(m)H_(2m−3), aryl, heteroaryl, alkylsulfide defined as CH₃(CH₂)_(n)—S—, imidoyl defined as R₃C(═NH)—,hemiacetal defined as R₄CH(OH)—S— and hydrogen, wherein R₁ and R₂ asdefined above can be unsubstituted or substituted. R₃ is hydrogen,alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylaryl, heteroaryl, orheterocyclyl, any of which can be substituted or unsubstituted; R₄ isCCl₃ or COOH. In addition, x is 0-16, z is preferably 0-5, morepreferably 0-3, n is 0-10 and m is 2-10. Suitable —[C(R₁)(R₂)]_(z)—groups include, without limitation, —CH₂, —CH(CH₃), —C(CH₃)₂, —CH(C₆H₅)and —CH(pyridine).

Also in this embodiment, a metal or metal salt can be added to one orboth sulfhydryls through a bond in which a metal or metal salt forms acovalent or coordination or chelated complex with the thiol group(s) ofthe lipoic acid molecule. Such metals include, platinum, nickel, silver,rhodium, cadmium, gold, palladium or cobalt. Metal salts include, forexample, platinum bromide, platinum chloride, platinum iodide, nickelborate, nickel boride, nickel bromide, nickel chloride, nickel iodide,nickel fluoride, silver bromate, silver bromide, silver chloride, silverfluoride, silver iodide, rhodium chloride, cadmium bromide, cadmiumchloride, cadmium fluoride, cadmium iodide, gold bromide, gold chloride,gold iodide, cobalt bromide, cobalt bromide, cobalt chloride, cobaltfluoride, cobalt iodide, palladium chloride, palladium iodide, andpalladium bromide. Such salts include various metal oxidation statessuch as, for example, platinum (II) chloride and platinum (IV) chloride.In general, the structure of the lipoic acid-metal complex describedherein is likely to be (metal)_(m) (lipoic acid)_(n) where m and n areboth one or where m is one and n is two.

Regardless of whether the lipoic acid derivative is of formula (I) orformula (II), pharmaceutical formulations of the present invention mayinclude lipoic acid derivatives in which one or both of the thiols havebeen replaced with a selenium molecule, a sulfur analog, or in which oneor both of the thiols have been oxidized to sulfate or related groups.

In particularly preferred embodiments of the present invention, thelipoic acid derivative is one selected from the following:

or a salt thereof (if not already in salt form).

Certain compounds described herein may exist in particular geometric orstereoisomeric forms. The present invention contemplates all suchcompounds, including cis- and trans-isomers, R- and S-enantiomers,diastereomers, (D)-isomers, (L)-isomers, racemic mixtures thereof,diastereomeric mixtures thereof, and other mixtures thereof, as fallingwithin the scope of the invention. Further, unless specificallyindicated otherwise, generic chemical structures presented herein areintended to encompass all geometric or stereoisomeric forms.

When the at least one lipoic acid derivative is a salt, it may benecessary to perform ion exchange in order to achieve ion pairing inaccordance with the invention. However, if a weak salt is used, an ionpairing agent such as triethanolamine could displace the anion withoutthe need for ion exchange.

Typically the at least one lipoic acid derivative is present in apharmaceutical formulation of the present invention in a therapeuticallyeffective amount. The pharmaceutical formulation of the presentinvention may contain a unit dose or multiple doses of the lipoic acidderivative. A “therapeutically effective amount” is intended to mean theamount of a lipoic acid derivative that, when administered to a subjectin need thereof, is sufficient to effect treatment for (or prevent)disease conditions characterized by disease cells that are sensitive tolipoic acid derivatives. The amount of a given lipoic acid derivativethat will be therapeutically effective will vary depending upon factorssuch as the disease condition and the severity thereof, the identity ofthe subject in need thereof, etc., which amount may be routinelydetermined by those of ordinary skill in the art. Importantly, thequantity of lipoic acid derivative in a unit dose should be sufficientto inhibit or kill tumor cells while leaving normal cells substantiallyunharmed. The at least one lipoic acid derivative is preferably presentin a pharmaceutical formulation of the present invention in an amount toprovide from about 0.001 mg/m² to about 10 g/m², more preferably about0.01 mg/m² to about 5 g/m², still more preferably from about 0.25 mg/m²to about 3 g/m², and most preferably from about 20 mg/m² to about 500mg/m² of the at least one lipoic acid derivative per dose. In certainother embodiments, the at least one lipoic acid derivative is present ina pharmaceutical formulation of the present invention in an amount toprovide from about 20 mg/m² to about 2500 mg/m², about 300 mg/m² toabout 700 mg/m², about 400 mg/m² to about 600 mg/m², about 380 mg/m² toabout 450 mg/m², about 410 mg/m² to about 430 mg/m², about 500 mg/m² toabout 700 mg/m², about 550 mg/m² to about 650 mg/m², or about 580 mg/m²to about 600 mg/m² of the at least one lipoic acid derivative per dose.

Importantly, the pharmaceutical formulations of the present inventionincludes at least one ion pairing agent. As used herein, “ion pairingagent” refers to any agent which is capable of forming a “salt bridge”or an “ion pair” with a given lipoic acid derivative. As used herein,“salt bridge” or “ion pair” refers to not only a salt (weak or strong)formed between an ion pairing agent and a given lipoic acid derivative,but also to other ionic associations (weak or strong) that do not riseto the level of actual salt formation between an ion pairing agent and agiven lipoic acid derivative. Without being bound by theory, it isbelieved that an ion pairing agent such as triethanolamine forms a saltbridge, i.e., forms a salt in situ, with a lipoic acid derivative suchas bis-benzyl lipoate, which then enables the lipoic acid deriviative toachieve its cell kill effect in vivo.

Ion pairing agents particularly suitable for use in the presentinvention include, without limitation, tertiary amines such astriethanolamine and polyethyleneimine, other amines such asdiethanolamine, monoethanolamine, mefenamic acid and tromethamine, andcombinations thereof. A preferred ion pairing agent is triethanolamine.In certain embodiments, the ion pairing agent is an organic Bronstedbase. In certain other embodiments, the ion pairing agent is an aminecompound. In certain preferred embodiments, the ion pairing agent is amonoalkylamine, dialkylamine, trialkylamine, amino-substituted aliphaticalcohol, hydroxymonoalkylamine, hydroxydialkylamine,hydroxytrialkylamine, amino-substituted heteroaliphatic alcohol,alkyldiamine, substituted alkyldiamine, or optionally substitutedheteroaryl group containing at least one ring nitrogen atom.

Additional ion pairing agents suitable for use in this invention includepolymer-conjugated ion pairing agents which employ, without limitation,polyethylene glycol, polyethyleneimine, polyglutamic acid andsugar-based polymers such as dextrans in combination with any of theabove-noted ion pairing agents or any other known ion pairing agent.Still further ion pairing agents can be selected with guidance fromHandbook of Pharmaceutical Salts: Properties, Selection and Use, IUPAC,Wiley-VCH, P. H. Stahl, ed., the entire disclosure of which isincorporated by reference herein. Ion pairing agents of particular notetherein include, without limitation, those listed in Table 5, p. 342,i.e., ammonia, L-arginine, benethamine benzathine, betaine, calciumhydroxide, choline, deanol, diethanolamine(2,2′-iminobis(ethanol)),diethylamine, 2-(diethylamino)-ethanol, ethanolamine, ethylenediamine,N-methyl-glucamine, hydrabamine, 1H-imidazole, lysine, magnesiumhydroxide, 4-(2-hydroxyethyl)-morpholine, piperazine, potassiumhydroxide, 1-(2-hydroxyethyl)-pyrrolidine, sodium hydroxide,triethanolamine (2,2′,2″-nitrilotris(ethanol)), tromethamine, and zinchydroxide. In certain other embodiments, the ion pairing agent isdiisopropanolamine, 3-amino-1-propanol, meglumine, morpholine, pyridine,niacinamide, tris(hydroxymethyl)aminomethane,2-((2-dimethylamino)ethoxy)ethanol, 2-(dimethylamino)ethanol,1-(2-hydroxyethyl)pyrrolidine, or ammonium hydroxide. In certain otherembodiments, the ion pairing agent is an alkali metal hydroxide oralkaline earth metal hydroxide, such as, for example, cesium hydroxide.

The ion pairing agent may be hydrophilic or hydrophobic (such asacylated triethanolamine). Typically the ion pairing agent is present inan amount sufficient to achieve substantial solubility of the at leastone lipoic acid derivative in a solvent suitable for intravenousadministration, which is most preferably an aqueous medium. Preferablythe ion pairing agent and lipoic acid derivative are present in a molarratio ranging from about 1000:1 to about 1:1000, more preferably fromabout 500:1 to about 1:500, still more preferably from about 50:1 toabout 1:50, still further more preferably from about 20:1 to about 1:20,and most preferably of about 1:1. In certain other embodiments, themolar ratio of ion pairing agent to lipoic acid derivative is about 1:1to about 10:1, or about 5:1 to about 10:1, or about 8:1.

Yet another embodiment of the present invention is directed to an ionpair, be it a true salt or some other lesser ionic association,consisting of (a) at least one lipoic acid derivative and (b) an ionpairing agent. In a highly preferred embodiment, the ion pair consistsof bis-benzyl lipoate and triethanolamine. The present inventionincludes all ion pairs, whether in situ as formed or isolated by someconventional method. All of the details regarding amounts of (a) and (b)and possible materials suitable for use are the same as those set forthin the general description above. Further, in certain embodiments, theinvention provides an ion pair consisting of, or in certain instancesconsisting essentially of:

-   -   (a) at least one ion pairing agent selected from the group        consisting of triethanolamine, polyethyleneimine,        diethanolamine, monoethanolamine, mefenamic acid, tromethamine,        ethanolamine, diethanolamine, ethylenediamine, lysine, and        diethylamine; and    -   (b) at least one lipoic acid derivative represented by Formula        (I):

wherein R₁ and R₂ are independently selected from the group consistingof acyl defined as R₃C(O)—, alkyl defined as C_(n)H_(2n+1), alkenyldefined as C_(m)H_(2m−1), alkynyl defined as C_(m)H_(2m−3), aryl,heteroaryl, alkyl sulfide defined as CH₃(CH₂)_(n)—S—, imidoyl defined asR₃C(═NH)—, hemiacetal defined as R₄CH(OH)—S—, and hydrogen provided thatat least one of R₁ and R₂ is not hydrogen;

wherein R₁ and R₂ as defined above can be unsubstituted or substituted;

wherein R₃ is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl,alkylaryl, heteroaryl, or heterocyclyl, any of which can be substitutedor unsubstituted;

wherein R₄ is CCl₃ or COOH; and

wherein x is 0-16, n is 0-10 and m is 2-10.

In certain embodiments, the ion pairing agent is a monoalkylamine,dialkylamine, amino-substituted aliphatic alcohol,hydroxymonoalkylamine, hydroxydialkylamine, hydroxytrialkylamine,amino-substituted heteroaliphatic alcohol, alkyldiamine, substitutedalkyldiamine, optionally substituted heteroaryl compound containing atleast one ring nitrogen atom. In certain other embodiments, the ionpairing agent is a monoalkylamine or dialkylamine. In certain otherembodiments, the ion pairing agent is amino-substituted aliphaticalcohol, hydroxymonoalkylamine, hydroxydialkylamine,hydroxytrialkylamine, or amino-substituted heteroaliphatic alcohol. Incertain other embodiments, the ion pairing agent is an alkyldiamine,substituted alkyldiamine, or optionally substituted heteroaryl compoundcontaining at least one ring nitrogen atom. In certain embodiments, theat least one ion pairing agent is triethanolamine. In certainembodiments, the at least one lipoic acid derivative is:

In certain embodiments, the at least one lipoic acid derivative isbis-benzyl lipoate and the at least one ion pairing agent istriethanolamine.II. Pharmaceutical Formulations Comprising a Lipoic Acid Derivative andIon Pairing Agent

One aspect of the present invention provides pharmaceutical formulationscomprising a lipoic acid derivative and an ion pairing agent. The lipoicacid derivative and ion pairing agent form an ion pair. Thepharmaceutical formulations can optionally include a pharmaceuticallyacceptable diluent. In particular, when a pharmaceutical formulationsuitable for, e.g., intravenous administration is desired, a suitablediluent would be employed. Any conventional aqueous or polar aproticsolvent is suitable for use in the present invention. Suitablepharmaceutically acceptable diluents include, without limitation,saline, a sugar solution, alcohols such as ethyl alcohol, methanol andisopropyl alcohol, polar aprotic solvents such as dimethylformamide(DMF), dimethylsulfoxide (DMSO) and dimethylacetamide (DMA), andcombinations thereof. A preferred pharmaceutically acceptable diluent isa dextrose solution, more preferably a dextrose solution containing fromabout 2.5% to about 10%, more preferably about 5%, dextrose by weight.The pharmaceutically acceptable diluent is typically employed in anon-homolysis generating amount; one of ordinary skill in the art canreadily determine an amount of diluent suitable for use in apharmaceutical formulation according to the present invention.

In a highly preferred embodiment of the present invention, thepharmaceutical formulation comprises bis-benzyl lipoate, triethanolamineand a dextrose solution containing about 5% dextrose by weight.

The pharmaceutical formulations of the present invention may optionallyinclude at least one other pharmaceutically acceptable additive.Suitable additives include, without limitation, solvents, diluents,surfactants, solubilizers, preservatives, buffers, and combinationsthereof, as well as any other additives particularly suited for use inparenteral administration forms. It is well within the skill of one ofordinary skill in the art to determine suitable amounts of these otherpharmaceutically acceptable additives. Solvents particularly suitablefor use herein include benzyl alcohol, dimethylamine, isopropyl alcoholand combinations thereof; one of ordinary skill in the art would readilyrecognize that it may be desirable to first dissolve the at least onelipoic acid derivative in a suitable solvent and then to dilute thesolution into an ion pairing agent and finally to dilute with a diluent.

The pharmaceutical formulations of the present invention can be preparedaccording to conventional formulation techniques. For example, a stocksolution of the at least one lipoic acid derivative and the ion pairingagent can be prepared according to conventional techniques and thendiluted as desired by a pharmaceutically acceptable diluent.

The pharmaceutical formulations of the present invention are liquidpreparations such as sterile parenteral solutions. The pharmaceuticalformulations of the present invention may be contained in any suitablevessel such as a vial or ampoule and are suitable for administration viaone of several routes including, without limitation, intravenous,intramuscular, subcutaneous, intradermally, intraperitoneal,intrathoracic, intrapleural, intrauterine or intratumor.

The pharmaceutical formulations generally described above will be moreparticularly described with reference to the particular embodimentsbelow. The embodiments described below are meant to more particularlydescribe various aspects of the invention and should not be construed aslimiting the invention.

Accordingly, one aspect of the invention provides a pharmaceuticalformulation comprising:

(a) a pharmaceutically acceptable diluent; and

(b) an ion pair formed by a lipoic acid derivative and an ion pairingagent; wherein the lipoic acid derivative is a compound of Formula Irepresented by:

wherein R₁ and R₂ are independently selected from the group consistingof acyl defined as R₃C(O)—, alkyl defined as C_(n)H_(2n+1), alkenyldefined as C_(m)H_(2m−1), alkynyl defined as C_(m)H_(2m−3), aryl,heteroaryl, alkyl sulfide defined as CH₃(CH₂)_(n)—S—, imidoyl defined asR₃C(═NH)—, hemiacetal defined as R₄CH(OH)—S—, and hydrogen provided thatat least one of R₁ and R₂ is not hydrogen;

wherein R₁ and R₂ as defined above can be unsubstituted or substituted;

wherein R₃ is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl,alkylaryl, heteroaryl, or heterocyclyl, any of which can be substitutedor unsubstituted;

wherein R₄ is CCl₃ or COOH; and

wherein x is 0-16, n is 0-10 and m is 2-10.

In certain embodiments, R₁ and R₂ are acetyl. In certain otherembodiments, R₁ and R₂ are benzoyl. In certain other embodiments, thearyl is benzyl. In certain other embodiments, R₁ and R₂ are benzyl. Incertain embodiments, the lipoic acid derivative is6,8-bis(benzylthio)octanoic acid, which is represented by the followingformula:

In certain embodiments, the ion pair is present in an amount that istherapeutically effective for treating cancer in a subject. In certainembodiments, the ion pair is present in an amount that istherapeutically effective for treating a microbial infection in asubject. In certain embodiments, the ion pair is present in an amount toprovide from about 0.001 mg/m² to about 10 g/m² of the lipoic acidderivative upon administration of the pharmaceutical formulation to apatient.

In certain embodiments, the ion pairing agent is an organic Bronstedbase compound. In certain embodiments, the ion pairing agent is an aminecompound. In certain embodiments, the ion pairing agent is amonoalkylamine, dialkylamine, trialkylamine, amino-substituted aliphaticalcohol, hydroxymonoalkylamine, hydroxydialkylamine,hydroxytrialkylamine, amino-substituted heteroaliphatic alcohol,alkyldiamine, substituted alkyldiamine, or an optionally substitutedheteroaryl compound containing at least one ring nitrogen atom. Incertain other embodiments, the ion pairing agent is a monoalkylamine,dialkylamine, or trialkylamine. In certain other embodiments, the ionpairing agent is amino-substituted aliphatic alcohol,hydroxymonoalkylamine, hydroxydialkylamine, hydroxytrialkylamine, oramino-substituted heteroaliphatic alcohol. In certain other embodiments,the ion pairing agent is an alkyldiamine, substituted alkyldiamine, oroptionally substituted heteroaryl compound containing at least one ringnitrogen atom. In certain other embodiments, the ion pairing agent is analkali metal hydroxide or an alkaline earth metal hydroxide. In certainother embodiments, the ion pairing agent is triethanolamine,polyethyleneimine, diethanolamine, monoethanolamine, mefenamic acid,tromethamine or a combination thereof. In certain other embodiments, theion pairing agent is triethanolamine. In certain other embodiments, theion pairing agent is ethanolamine, diethanolamine, ethylenediamine,lysine, diethylamine, or triethylamine. In certain other embodiments,the ion pairing agent is sodium hydroxide, potassium hydroxide, orammonia. In certain other embodiments, the ion pairing agent isdiisopropanolamine, 3-amino-1-propanol, meglumine, morpholine, pyridine,niacinamide, tris(hydroxymethyl)aminomethane,2-((2-dimethylamino)ethoxy)ethanol, 2-(dimethylamino)ethanol,1-(2-hydroxyethyl)pyrrolidine, triisopropanolamine, ammonium hydroxide,or cesium hydroxide. In certain other embodiments, the ion pairing agentis piperazine.

It is further contemplated that the ion pairing agent may be a polymer.In particular, a variety of polymers are contemplated for use as the ionpairing agent, including linear polymers and branched polymers. Incertain embodiments, the polymer has a weight average molecular weightof about 200 g/mol to about 300,000 g/mol, about 200 g/mol to about200,000 g/mol, about 200 g/mol to about 100,000 g/mol, about 1,000 g/molto about 100,000 g/mol, about 5,000 g/mol to about 50,000 g/mol, orabout 50,000 g/mol to about 75,000 g/mol. In certain embodiments, thepolymer is a polyethylene glycol, polyethyleneimine, or dextran. Incertain other embodiments, the polymer is a polyethyleneimine, dextran,or a polyethylene glycol having a weight average molecular weight ofabout 250 g/mol to about 100,000 g/mol. In certain embodiments, thepolyethylene glycol has a weight average molecular weight of about 250g/mol to about 40,000 g/mol, or about 500 g/mol to about 40,000 g/mol.

In certain embodiments, the ion pairing agent and lipoic acid derivativeare present in a mole ratio ranging from about 1000:1 to about 1:1000.In certain other embodiments, the ion pairing agent and lipoic acidderivative are present in a mole ratio ranging from about 1:1 to about10:1, or about 5:1 to about 10:1. In certain other embodiments, the moleratio of the ion pairing agent and lipoic acid derivative is about 6:1,7:1, 8:1 or 9:1. The various ion pairing agent embodiments describedabove are contemplated for use in forming ion pairs with all of thegeneric and specific lipoic acid derivatives described herein. Oneparticularly preferred specific lipoic acid derivate is6,8-bis(benzylthio)octanoic acid. In certain embodiments, the ion pairformed by the lipoic acid derivative and the ion pairing agent is asalt.

In certain embodiments, the diluent is selected from the groupconsisting of saline, a sugar solution, an alcohol, dimethylformamide,dimethylsulfoxide, dimethylacetamide and combinations thereof. Incertain embodiments, the diluent is a dextrose solution. In certainother embodiments, the dextrose solution contains an amount of dextroseranging from about 2.5% to about 10% by weight. In certain otherembodiments, the pharmaceutical formulation further comprises at leastone pharmaceutically acceptable additive selected from solvents,diluents, surfactants, solubilizers, preservatives, buffers, andcombinations thereof.

Another aspect of the invention provides a pharmaceutical formulationcomprising: bis-benzyl lipoate, and triethanolamine. In certainembodiments, the formulation further comprises a dextrose solutioncontaining about 5% dextrose by weight.

Another aspect of the invention provides an intravenous pharmaceuticalcomposition for treating cancer, comprising 6,8-bis(benzylthio)octanoicacid or an ion pair thereof in an amount effective for treating cancer,and a pharmaceutically acceptable aqueous diluent for solubilizing6,8-bis(benzylthio)octanoic acid or an ion pair thereof. In certainembodiments, the diluent comprises saline. In certain embodiments, thecomposition provides 6,8-bis(benzylthio)octanoic acid or an ion pairthereof in an amount sufficient to provide a patient with from about0.001 mg/m² to about 10 g/m² of 6,8-bis(benzylthio)octanoic acid perdose of the intravenous pharmaceutical composition. In certainembodiments, the composition provides 6,8-bis(benzylthio)octanoic acidor an ion pair thereof in an amount sufficient to provide a patient withfrom about 20 mg/m² to about 2500 mg/m² of 6,8-bis(benzylthio)octanoicacid per dose of the intravenous pharmaceutical composition. In certainembodiments, the composition provides 6,8-bis(benzylthio)octanoic acidor an ion pair thereof in an amount sufficient to provide a patient withfrom about 20 mg/m² to about 500 mg/m² of 6,8-bis(benzylthio)octanoicacid per dose of the intravenous pharmaceutical composition. In certainembodiments, the composition provides 6,8-bis(benzylthio)octanoic acidor an ion pair thereof in an amount sufficient to provide a patient withfrom about 300 mg/m² to about 700 mg/m², about 400 mg/m² to about 600mg/m², about 380 mg/m² to about 450 mg/m², about 410 mg/m² to about 430mg/m², about 500 mg/m² to about 700 mg/m², about 550 mg/m² to about 650mg/m², or about 580 mg/m² to about 600 mg/m² of lipoic acid derivative,e.g., 6,8-bis(benzylthio)octanoic acid, per dose of the intravenouspharmaceutical composition. In certain embodiments, the compositioncomprises 6,8-bis(benzylthio)octanoic acid in the form of an ion pair.In certain other embodiments, the composition comprises6,8-bis(benzylthio)octanoic acid in the form of an ion pair withtriethanolamine.

In certain embodiments, the pharmaceutical formulations and compositionsdescribed herein contain less than 15% by weight impurities, less than10% by weight impurities, less than 5% by weight impurities, less than2% by weight impurities, or less than 1% by weight impurities. In apreferred embodiment, the pharmaceutical formulations and compositionsdescribed herein contain less than 2% by weight impurities.

In certain other embodiments, the lipoic acid derivative used to preparethe pharmaceutical formulations and compositions described herein has apurity of at least 35% by weight, at least 85% by weight, at least 90%by weight, at least 95% by weight, at least 98% by weight, or at least99% by weight. In a preferred embodiment, the lipoic acid derivativeused to prepare the pharmaceutical formulations and compositionsdescribed herein has a purity of at least at least 99% by weight.

In certain embodiments, the pharmaceutical formulations and compositionsdescribed herein are characterized in that exposing a population ofA2780 tumor cells to the pharmaceutical formulation or composition at aconcentration of a 200 μM with respect to the ion pair results in thedeath of greater than 70% of the tumor cells in the population (i.e.,results in less than 30% tumor cell viability). Illustrative proceduresfor conducting an experiment to determine the degree of tumor cell deathare described in Example 2 below. In certain other embodiments, thepharmaceutical formulations and compositions described herein arecharacterized in that exposing a population of A2780 tumor cells to thepharmaceutical formulation or composition at a concentration of a 300 μMwith respect to the ion pair results in the death of greater than 95% ofthe tumor cells in the population (i.e., results in less than 5% tumorcell viability). In certain other embodiments, the pharmaceuticalformulations and compositions described herein are characterized in thatexposing a population of A2780 tumor cells to the pharmaceuticalformulation at a concentration of a 400 μM with respect to the ion pairresults in the death of greater than 99% of the tumor cells in thepopulation (i.e., results in less than 1% tumor cell viability).

In certain embodiments, the pharmaceutical formulations and compositionsdescribed herein are characterized in that administering saidpharmaceutical formulation or composition to a subject suffering fromcancer, the pharmaceutical formulation or composition administered at anacceptably tolerated daily dosage for a period of two weeks, results ina 5% reduction in tumor volume. In certain embodiments, thepharmaceutical formulations and compositions described herein arecharacterized in that administering said pharmaceutical formulation orcomposition to a subject suffering from cancer, the pharmaceuticalformulation or composition administered at an acceptably tolerated dailydosage for a period of two weeks, results in a 25%, 35%, 40%, 45%, 50%,or 60% reduction in tumor volume. In certain embodiments, the cancer isa cancer of the lung, liver, uterus, cervix, bladder, kidney, colon,breast, prostate, ovary, or pancreas. In certain embodiments, thesubject suffering from cancer is a murine subject. In certain otherembodiments, subject suffering from cancer is a human subject. The term“acceptably tolerated dose” is art-recognized and refers to a dosehaving an associated probability of toxicity as close as possible toacceptable toxicity. Acceptable toxicity means a toxicity falling withina predefined measure of adverse events related to the protocoltreatment. See, for example, Handbook of Statistics in ClinicalOncology, 2nd ed., passim. Crowley J and Ankerst D P (eds.), 2006. NewYork: Chapman & Hall/CRC.)

In certain embodiments, the pharmaceutical formulations and compositionsdescribed herein are characterized in that administering saidpharmaceutical formulation or composition to a subject suffering fromcancer, the pharmaceutical formulation or composition administered at atherapeutically effective daily dosage for a period of two weeks,results in a 5% reduction in tumor volume. In certain embodiments, thepharmaceutical formulations and compositions described herein arecharacterized in that administering said pharmaceutical formulation orcomposition to a subject suffering from cancer, the pharmaceuticalformulation or composition administered at a therapeutically effectivefor a period of two weeks, results in a 25%, 35%, 40%, 45%, 50%, or 60%reduction in tumor volume. In certain embodiments, the cancer is acancer of the lung, liver, uterus, cervix, bladder, kidney, colon,breast, prostate, ovary, or pancreas. In certain embodiments, thesubject suffering from cancer is a murine subject. In certain otherembodiments, subject suffering from cancer is a human subject.

In certain embodiments, the pharmaceutical formulations and compositionsdescribed herein are characterized in that administering saidpharmaceutical formulation or composition to a subject suffering fromcancer, the pharmaceutical formulation or composition administered at anacceptably tolerated daily dosage for a period of two weeks, results ina 5% reduction in the number of cancer cells in said subject. In certainembodiments, the pharmaceutical formulations and compositions describedherein are characterized in that administering said pharmaceuticalformulation or composition to a subject suffering from cancer, thepharmaceutical formulation or composition administered at an acceptablytolerated daily dosage for a period of two weeks, results in a 25%, 35%,40%, 45%, 50%, or 60% reduction in the number of cancer cells in saidsubject. In certain embodiments, the cancer is a cancer of the lung,liver, uterus, cervix, bladder, kidney, colon, breast, prostate, ovary,or pancreas.

In certain embodiments, the pharmaceutical formulations and compositionsdescribed herein are characterized in that administering saidpharmaceutical formulation or composition to a subject suffering fromcancer, the pharmaceutical formulation or composition administered at atherapeutically effective daily dosage for a period of two weeks,results in a 5% reduction in the number of cancer cells in said subject.In certain embodiments, the pharmaceutical formulations and compositionsdescribed herein are characterized in that administering saidpharmaceutical formulation or composition to a subject suffering fromcancer, the pharmaceutical formulation or composition administered at atherapeutically effective daily dosage for a period of two weeks,results in a 25%, 35%, 40%, 45%, 50%, or 60% reduction in the number ofcancer cells in said subject. In certain embodiments, the cancer is acancer of the lung, liver, uterus, cervix, bladder, kidney, colon,breast, prostate, ovary, or pancreas.

A number of known methods can be used to assess the volume of a tumor.Non-limiting examples of such methods include imaging methods (e.g.,computed tomography (CT), magnetic resonance imaging (MRI), ultrasound,X-ray imaging, mammography, PET scans, radionuclide scans, bone scans),visual methods (e.g., colonoscopy, bronchoscopy, endoscopy), physicalexamination (e.g., prostate examination, breast examination, lymph nodesexamination, abdominal examination, rectal examination, generalpalpation), blood tests (e.g., prostate specific antigen (PSA) test,carcinoembryonic antigen (CEA) test, cancer antigen (CA)-125 test,alpha-fetoprotein (AFP), liver function tests), bone marrow analyses(e.g., in cases of hematological malignancies), histopathology,cytology, and flow cytometry

In yet other aspects, the invention provides a pharmaceuticalformulation comprising:

(a) at least one lipoic acid derivative or salt thereof; and

(b) at least one ion pairing agent,

wherein the lipoic acid derivative and the ion pairing agent form an ionpair.

In certain embodiments, the at least one lipoic acid derivative has theformula (I):

wherein R₁ and R₂ are independently selected from the group consistingof acyl defined as R₃C(O)—, alkyl defined as C_(n)H_(2n+1), alkenyldefined as C_(m)H_(2m−1), alkynyl defined as C_(m)H_(2m−3), aryl,heteroaryl, alkyl sulfide defined as CH₃(CH₂)—S—, imidoyl defined asR₃C(═NH)—, hemiacetal defined as R₄CH(OH)—S—, and hydrogen provided thatat least one of R₁ and R₂ is not hydrogen; wherein R₁ and R₂ as definedabove can be unsubstituted or substituted; wherein R₃ is hydrogen,alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylaryl, heteroaryl, orheterocyclyl, any of which can be substituted or unsubstituted; whereinR₄ is CCl₃ or COOH; and wherein x is 0-16, n is 0-10 and m is 2-10. Incertain embodiments, R₁ and R₂ are acetyl groups. In certainembodiments, R₁ and R₂ are benzoyl groups. In certain embodiments, thealkenyl is selected from the group consisting of propenyl,2,3-dimethyl-2-butenyl and heptenyl. In certain embodiments, the alkynylis selected from the group consisting of acetylenyl, propynyl andoctynyl. In certain embodiments, the aryl is a benzyl or a benzylderivative. In certain embodiments, R₁ and R₂ are each a benzyl group.In certain embodiments, the alkyl is cyclopropyl. In certainembodiments, the alkenyl is cyclopentyl. In certain embodiments, the atleast one lipoic acid derivative has the formula (II):

wherein M is a metal chelate, —[C(R₁)(R₂)]_(z)— or other metal complex;wherein R₁ and R₂ are independently selected from the group consistingof acyl defined as R₃C(O)—, alkyl defined as C_(n)H_(2n+1), alkenyldefined as C_(m)H_(2m−1), alkynyl defined as C_(m)H_(2m−3), aryl,heteroaryl, alkyl sulfide defined as CH₃(CH₂)_(n)—S—, imidoyl defined asR₃C(═NH)—, hemiacetal defined as R₄CH(OH)—S—, and hydrogen; wherein R₁and R₂ as defined above can be unsubstituted or substituted; wherein R₃is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylaryl,heteroaryl, or heterocyclyl, any of which can be substituted orunsubstituted; wherein R₄ is CCl₃ or COOH; and wherein x is 0-16, z is0-5, n is 0-10 and m is 2-10.

In certain embodiments, the at least one lipoic acid derivative ispresent in a therapeutically effective amount. In certain embodiments,the at least one lipoic acid derivative is present in an amount toprovide from about 0.001 mg/m² to about 10 g/m² of the at least onelipoic acid derivative. In certain embodiments, the at least one ionpairing agent is selected from the group consisting of triethanolamine,polyethyleneimine, diethanolamine, monoethanolamine, mefenamic acid,tromethamine and combinations thereof. In certain embodiments, the atleast one ion pairing agent is triethanolamine. In certain embodiments,the at least one ion pairing agent is a polymer-conjugated ion pairingagent. In certain embodiments, the at least one ion pairing agent andthe at least one lipoic acid derivative is present in a ratio rangingfrom about 1000:1 to about 1:1000. In certain embodiments, thepharmaceutical formulation further comprises a pharmaceuticallyacceptable diluent. In certain embodiments, the diluent is selected fromthe group consisting of saline, a sugar solution, an alcohol,dimethylformamide, dimethylsulfoxide, dimethylacetamide and combinationsthereof. In certain embodiments, the diluent is a dextrose solution. Incertain embodiments, the dextrose solution contains an amount ofdextrose ranging from about 2.5% to about 10% by weight. In certainembodiments, the pharmaceutical formulation further comprises at leastone pharmaceutically acceptable additive selected from solvents,diluents, surfactants, solubilizers, preservatives, buffers, andcombinations thereof. In certain embodiments, the ion pair is a salt.

In yet other aspects, the invention provides a pharmaceuticalformulation comprising bis-benzyl lipoate; and triethanolamine. Incertain embodiments, the pharmaceutical formulation further comprises adextrose solution containing about 5% dextrose by weight.

In yet other aspects, the invention provides an ion pair consisting of:at least one lipoic acid derivative; and at least one ion pairing agent.In certain embodiments, the at least one lipoic acid derivative has theformula (I):

wherein R₁ and R₂ are independently selected from the group consistingof acyl defined as R₃C(O)—, alkyl defined as C_(n)H_(2n+1), alkenyldefined as C_(m)H_(2m−1), alkynyl defined as C_(m)H_(2m−3), aryl,heteroaryl, alkyl sulfide defined as CH₃(CH₂)_(n)—S—, imidoyl defined asR₃C(═NH)—, hemiacetal defined as R₄CH(OH)—S—, and hydrogen provided thatat least one of R₁ and R₂ is not hydrogen; wherein R₁ and R₂ as definedabove can be unsubstituted or substituted; wherein R₃ is hydrogen,alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylaryl, heteroaryl, orheterocyclyl, any of which can be substituted or unsubstituted; whereinR₄ is CCl₃ or COOH; and wherein x is 0-16, n is 0-10 and m is 2-10. Incertain embodiments, the at least one lipoic acid derivative has theformula (II):

wherein M is a metal chelate, —[C(R₁)(R₂)]_(z)— or other metal complex;wherein R₁ and R₂ are independently selected from the group consistingof acyl defined as R₃C(O)—, alkyl defined as C_(n)H_(2n+1), alkenyldefined as C_(m)H_(2m−1), alkynyl defined as C_(m)H_(2m−3), aryl,heteroaryl, alkyl sulfide defined as CH₃(CH₂)_(n)—S—, imidoyl defined asR₃C(═NH)—, hemiacetal defined as R₄CH(OH)—S—, and hydrogen; wherein R₁and R₂ as defined above can be unsubstituted or substituted; wherein R₃is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylaryl,heteroaryl, or heterocyclyl, any of which can be substituted orunsubstituted; wherein R₄ is CCl₃ or COOH; and wherein x is 0-16, z is0-5, n is 0-10 and m is 2-10. In certain embodiments, wherein the atleast one ion pairing agent is selected from the group consisting oftriethanolamine, polyethyleneimine, diethanolamine, monoethanolamine,mefenamic acid, tromethamine and combinations thereof. In certainembodiments, the at least one ion pairing agent is a polymer-conjugatedion pairing agent. In certain embodiments, the at least one ion pairingagent and the at least one lipoic acid derivative is present in a ratioranging from about 1000:1 to about 1:1000. In certain embodiments, theion pair is a salt. In certain embodiments, the at least one lipoic acidderivative is bis-benzyl lipoate and the at least one ion pairing agentis triethanolamine.III. Therapeutic Applications

Another aspect of the invention is directed to a method of treating adisease characterized by disease cells that are sensitive to lipoic acidderivatives comprising administering to a patient in need thereof apharmaceutical formulation described herein. In yet other aspects, theinvention provides a method of preventing a disease characterized bydisease cells that are sensitive to lipoic acid derivatives comprisingadministering to a patient in need thereof a pharmaceutical formulationdescribed herein.

In certain embodiments of these methods, pharmaceutical formulations oflipoic acid derivatives may be used to prevent or inhibit diseasesinvolving altered or distinct cellular PDC activity, i.e., diseasescharacterized by disease cells that are sensitive to lipoic acidderivatives. Cells with appropriately altered or deranged energymetabolism, i.e., altered PDC activity, are particularly targeted andkilled, while surrounding healthy tissues remain unharmed by the lipoicacid derivative. The skilled artisan can readily identify diseaseshaving altered PDC activity. Alternatively, the skilled artisan canreadily screen the disease of interest for sensitivity to lipoic acidderivatives.

In another aspect, the invention provides a method of treating orpreventing a disease characterized by disease cells that are sensitiveto lipoic acid derivatives, the method comprising administering to apatient in need thereof a pharmaceutical formulation described herein totreat or prevent said disease. In certain embodiments, the disease isselected from the group consisting of carcinoma, sarcoma, myeloma,lymphoma, leukemia and mixed types thereof. In certain embodiments, thedisease is a microbial infection. In certain embodiments, the microbialinfection is a bacterial infection, such as an infection by anActinomyces, a Campylobacter (e.g., Campylobacter jejuni), anEscherichia (e.g., Escherichia coli), a Leptospira, a Pseudomonas (e.g.,Pseudomonas aeruginosa), a Shigella (e.g., Shigella boydii), aStaphylococcus (e.g., Staphylococcal aureus), or a Streptococcus (e.g.,Streptococcus pneumoniae) bacterium. In certain other embodiments, themicrobial infection is a yeast infection (e.g., a Candida) or fungalinfection (e.g., a Cryptococcus). In certain other embodiments, themicrobial infection is a eukaryotic infection, e.g., by Cryptosporidium,Giardia, Leishmania, Neospora, Plasmodia, Toxoplasma, Trichomonas, orTrypanosoma.

Further aspects of the invention provide a method of treating cancer ina patient, comprising administering to a patient in need thereof atherapeutically effective amount of a pharmaceutical compositioncomprising 6,8-bis(benzylthio)octanoic acid or an ion pair thereof, anda pharmaceutically acceptable diluent for solubilizing6,8-bis(benzylthio)octanoic acid or an ion pair thereof. In certainembodiments, the diluent comprises saline. In certain embodiments, thepharmaceutical composition is an intravenous pharmaceutical compositionthat is administered to the patient intravenously. In certainembodiments, the composition provides 6,8-bis(benzylthio)octanoic acidor an ion pair thereof in an amount sufficient to provide a patient withfrom about 0.001 mg/m² to about 10 g/m² of 6,8-bis(benzylthio)octanoicacid per dose of the intravenous pharmaceutical composition. In certainother embodiments, the composition provides 6,8-bis(benzylthio)octanoicacid or an ion pair thereof in an amount sufficient to provide a patientwith from about 20 mg/m² to about 2500 mg/m² of6,8-bis(benzylthio)octanoic acid per dose of the intravenouspharmaceutical composition. In certain embodiments, the compositionprovides 6,8-bis(benzylthio)octanoic acid or an ion pair thereof in anamount sufficient to provide a patient with from about 20 mg/m² to about500 mg/m² of 6,8-bis(benzylthio)octanoic acid per dose of theintravenous pharmaceutical composition. In certain embodiments, thecomposition provides 6,8-bis(benzylthio)octanoic acid or an ion pairthereof in an amount sufficient to provide a patient with from about 300mg/m² to about 700 mg/m², about 400 mg/m² to about 600 mg/m², about 380mg/m² to about 450 mg/m², about 410 mg/m² to about 430 mg/m², about 500mg/m² to about 700 mg/m², about 550 mg/m² to about 650 mg/m², or about580 mg/m² to about 600 mg/m² of 6,8-bis(benzylthio)octanoic acid perdose of the intravenous pharmaceutical composition.

In certain embodiments, the composition comprises6,8-bis(benzylthio)octanoic acid in the form of an ion pair. In certainother embodiments, the composition comprises 6,8-bis(benzylthio)octanoicacid in the form of an ion pair with triethanolamine.

In preferred embodiments of the methods of the present invention, thedisease treated or prevented includes cancer, such as carcinoma,sarcoma, myeloma, lymphoma, leukemia and mixed types thereof. Thepharmaceutical formulations of the present invention are effectiveagainst both primary and metastatic cancers and effective againstcancers of the, without limitation, lung, liver, uterus, cervix,bladder, kidney, colon, breast, prostate, ovary, and pancreas. In otherembodiments, the pharmaceutical formulations of the present inventioncan be used in the treatment of diseases associated with altered energymetabolism such as Alzheimer's disease, hyperproliferative diseases suchas psoriasis and other diseases such as diabetic neuropathy.

For certain therapeutic applications, a pharmaceutical formulation isadministered directly to a patient, typically in a unit dose form. Incertain methods of this invention, the pharmaceutical formulationcomprising the lipoic acid derivative may be administered via one ofseveral routes including, without limitation, intravenous,intramuscular, subcutaneous, intradermally, intraperitoneal,intrathoracic, intrapleural, intrauterine or intratumor. Those skilledin the art will recognize that the mode of administering the lipoic acidderivative depends on the type of cancer or symptom to be treated. Forexample, a preferred mode of administering the lipoic acid for treatmentof leukemia would involve intravenous administration. Likewise, thoseskilled in the art will also recognize that particular pharmaceuticallyacceptable additives will vary from pharmaceutical formulations suitablefor one administration mode to pharmaceutical formulations suitable foranother administration mode—the constant in all pharmaceuticalformulations regardless of intended mode of administration, however, isthe presence of an ion pair formed between the at least one lipoic acidderivative and the ion pairing agent.

By adapting the treatments described herein, the pharmaceuticalformulations of the present invention may also be used in methods fortreating diseases other than cancer, where the disease-causing cellsexhibit altered metabolic patterns. For example, eukaryotic pathogens ofhumans and other animals are generally much more difficult to treat thanbacterial pathogens because eukaryotic cells are so much more similar toanimal cells than are bacterial cells. Such eukaryotic pathogens includeprotozoans such as those causing malaria as well as fungal and algalpathogens. Because of the remarkable lack of toxicity of the lipoic acidderivatives used in the invention to normal human and animal cells andbecause many eukaryotic pathogens are likely to pass through life cyclestages in which their PDCs become sensitive to lipoic acid derivatives,the pharmaceutical formulations of the present invention can be used tokill bacterial PDCs.

In general, a suitable daily dose of a ion pair compound will be thatamount of the compound which is the highest tolerated dose and/or thelowest dose effective to produce a therapeutic effect. Suitable dosageformulations and methods of administering the agents are readilydetermined by those of skill in the art. Preferably, the ion paircompound is administered at about 0.01 mg/kg to about 200 mg/kg, morepreferably at about 0.1 mg/kg to about 100 mg/kg, even more preferablyat about 0.5 mg/kg to about 50 mg/kg, about 1 mg/kg to about 20 mg/kg,about 5 mg/kg to about 20 mg/kg, or about 10 mg/kg to about 30 mg/kg. Incertain other embodiments, the ion pair compound is administered atabout 30 mg/kg to about 125 mg/kg, at about 50 mg/kg to about 100 mg/kg,at about 60 mg/kg to about 90 mg/kg, at about 65 mg/kg to about 75mg/kg, at about 65 mg/kg to about 85 mg/kg, or at about 60 mg/kg toabout 75 mg/kg.

In certain embodiments, the methods described herein compriseadministering a therapeutically effective amount of the pharmaceuticalformulation to the patient at least two times per week for at least twoweeks. In certain embodiments, the methods described herein compriseadministering a therapeutically effective amount of the pharmaceuticalformulation to the patient one to three times per week for at least twoweeks. In certain embodiments, the methods described herein compriseadministering a therapeutically effective amount of the pharmaceuticalformulation to the patient two times per week for at least two weeks. Incertain other embodiments, the methods described herein compriseadministering a therapeutically effective amount of the pharmaceuticalformulation to the patient one to three times per week for a period oftwo weeks, then no further anti-cancer therapeutics are administered tothe patient for at least 3 days, at least 5 days, or at least 7 days. Incertain other embodiments, the methods described herein compriseadministering a therapeutically effective amount of the pharmaceuticalformulation to the patient two times per week for a period of two weeks,then no further anti-cancer therapeutics are administered to the patientfor at least 3 days, at least 5 days, or at least 7 days. In certainother embodiments, the methods described herein comprise administering atherapeutically effective amount of the pharmaceutical formulation tothe patient two times per week for a period of two weeks, then nofurther anti-cancer therapeutics are administered to the patient for atleast 1 week, and then the pharmaceutical formulation is administered tothe patient at least two times per week for a period of at least oneweek. In certain other embodiments, the methods described hereincomprise administering a therapeutically effective amount of thepharmaceutical formulation to the patient two times per week for aperiod of two weeks, then no further anti-cancer therapeutics areadministered to the patient for at least 1 week, and then this dosingregimine is repeated beginning by administering a therapeuticallyeffective amount of the pharmaceutical formulation to the patient twotimes per week for a period of two weeks.

Another aspect of the invention provides a method of inducing necroticdeath of a cancer cell. The method comprises administering to a cancercell an effective amount of a pharmaceutical formulation comprising (a)a pharmaceutically acceptable diluent; and (b) an ion pair formed by alipoic acid derivative of Formula I, and an ion pairing agent that is anorganic Bronsted base compound; wherein Formula I represented by:

wherein R₁ and R₂ are independently selected from the group consistingof acyl defined as R₃C(O)—, alkyl defined as C_(n)H_(2n+1), alkenyldefined as C_(m)H_(2m−1), alkynyl defined as C_(m)H_(2m−3), aryl,heteroaryl, alkyl sulfide defined as CH₃(CH₂)_(n)—S—, imidoyl defined asR₃C(═NH)—, hemiacetal defined as R₄CH(OH)—S—, and hydrogen provided thatat least one of R₁ and R₂ is not hydrogen;

wherein R₁ and R₂ as defined above can be unsubstituted or substituted;

wherein R₃ is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl,alkylaryl, heteroaryl, or heterocyclyl, any of which can be substitutedor unsubstituted;

wherein R₄ is CCl₃ or COOH; and

wherein x is 0-16, n is 0-10 and m is 2-10.

In certain embodiments, the method preferentially induces necrotic deathof the cancer cell. In particular, embodiments are contemplated wherethe method induces a greater proportion of cell death by necrosis, thanby apoptosis. In certain embodiments, the ion pairing agent is an aminecompound. In certain embodiments, the ion pairing agent is amonoalkylamine, dialkylamine, trialkylamine, amino-substituted aliphaticalcohol, hydroxymonoalkylamine, hydroxydialkylamine,hydroxytrialkylamine, amino-substituted heteroaliphatic alcohol,alkyldiamine, substituted alkyldiamine, or an optionally substitutedheteroaryl compound containing at least one ring nitrogen atom. Incertain other embodiments, the ion pairing agent is a monoalkylamine,dialkylamine, or trialkylamine. In certain other embodiments, the ionpairing agent is triethanolamine, polyethyleneimine, diethanolamine,monoethanolamine, mefenamic acid, tromethamine or a combination thereof.In certain other embodiments, the ion pairing agent is ethanolamine,diethanolamine, ethylenediamine, lysine, diethylamine, or triethylamine.In certain other embodiments, the ion pairing agent isdiisopropanolamine, 3-amino-1-propanol, meglumine, morpholine, pyridine,niacinamide, tris(hydroxymethyl)aminomethane,2-((2-dimethylamino)ethoxy)ethanol, 2-(dimethylamino)ethanol,1-(2-hydroxyethyl)pyrrolidine, triisopropanolamine, or ammoniumhydroxide. In certain other embodiments, the ion pairing agent istriethanolamine. In certain embodiments, the lipoic acid derivative inthe pharmaceutical composition is 6,8-bis(benzylthio)octanoic acid, andsaid ion pairing agent is triethanolamine.

Another aspect of the invention provides a method of inducing apoptoticdeath of a cancer cell. The method comprises administering to a cancercell an effective amount of a pharmaceutical formulation comprising (a)a pharmaceutically acceptable diluent; and (b) an ion pair formed by alipoic acid derivative of Formula I, and an ion pairing agent that is analkali metal hydroxide or an alkaline earth metal hydroxide; whereinFormula I represented by:

wherein R₁ and R₂ are independently selected from the group consistingof acyl defined as R₃C(O)—, alkyl defined as C_(n)H_(2n+1), alkenyldefined as C_(m)H_(2m−1), alkynyl defined as C_(m)H_(2m−3), aryl,heteroaryl, alkyl sulfide defined as CH₃(CH₂)_(n)—S—, imidoyl defined asR₃C(═NH)—, hemiacetal defined as R₄CH(OH)—S—, and hydrogen provided thatat least one of R₁ and R₂ is not hydrogen;

wherein R₁ and R₂ as defined above can be unsubstituted or substituted;

wherein R₃ is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl,alkylaryl, heteroaryl, or heterocyclyl, any of which can be substitutedor unsubstituted;

wherein R₄ is CCl₃ or COOH; and

wherein x is 0-16, n is 0-10 and m is 2-10.

In certain embodiments, the method preferentially induces apoptoticdeath of the cancer cell. In particular, embodiments are contemplatedwhere the method induces a greater proportion of cell death byapoptosis, than by necrosis. In certain embodiments, the ion pairingagent is sodium hydroxide, potassium hydroxide, cesium hydroxide,calcium hydroxide, or magnesium hydroxide. In certain other embodiments,the ion pairing agent is sodium hydroxide or potassium hydroxide. Incertain embodiments, the lipoic acid derivative in said pharmaceuticalcomposition is 6,8-bis(benzylthio)octanoic acid, and said ion pairingagent is sodium hydroxide.

Specific embodiments of the invention will now be demonstrated byreference to the following examples. It should be understood that theseexamples are disclosed solely by way of illustrating the invention andshould not be taken in any way to limit the scope of the presentinvention.

EXAMPLE 1

Bis-benzyl lipoate was provided in a concentrated form at aconcentration of 50 mg/mL dissolved in 1M triethanolamine (TEA). Thestability of the drug product was assessed by visual observation and byhigh-performance liquid chromatography (HPLC) assessment, performed atthe beginning and the end of the study. The physical appearance did notchange and the purity was found to be >99% pure, both at the beginningand the end of the study. The concentrated bis-benzyl lipoate solutionwas diluted to an appropriate concentration with 5% dextrose (D5W) toformulate 0.1, 1 and 10 mg/kg doses of bis-benzyl lipoate.

COMPARATIVE EXAMPLE 1

Bis-benzyl lipoate was dissolved to a concentration of 40 mg/mL in aconventional mixture of Tween 80 and ethanol (1:1 by volume ratio). Theconcentrated bis-benzyl lipoate solution was diluted to an appropriateconcentration with saline.

Testing

A study to assess the dose and dosing schedule effects on the anti-tumoractivity of bis-benzyl lipoate was undertaken. More specifically, thepharmaceutical formulations of bis-benzyl lipoate of Comparative Example1, i.e., bis-benzyl lipoate dissolved in 1:1 Tween 80:ethanol anddiluted with saline, were tested in mice with Human H-460 Non Small CellLung Carcinoma (NSCLC) xenograft. The pharmaceutical formulations wereadministered intraperitoneally (IP), given 1× or 3× weekly.Administration of bis-benzyl lipoate began when the average tumor sizeof the mice was ˜300 mm³. There were originally eight treatment groups,with seven mice in each group, investigating three doses (0.1, 1 and 10mg/kg) and two dosing schedules, as shown in Table 1 below.

TABLE 1 Original Treatment Groups. Dose of bis-benzyl Treatment Grouplipoate (mg/kg) Dose Schedule # of mice 1 0 (vehicle*) 1x weekly 7 2 0(vehicle*) 3x weekly 7 3 0.1 1x weekly 7 4 0.1 3x weekly 7 5 1 1x weekly7 6 1 3x weekly 7 7 10 1x weekly 7 8 10 3x weekly 7 *vehicle refers to aTween 80/ethanol/saline mixture

The results (as shown in FIGS. 1A and 1B) showed that bis-benzyl lipoatedid not induce any anti-tumor effects, when compared to vehicletreatment.

Next, the protocol was revised by subdividing each treatment group intotwo subgroups, as shown in Table 2 below. Specifically, both subgroupsof each treatment group were treated with the same dose of bis-benzyllipoate as in the original protocol; however, one of the two subgroupswas treated with a pharmaceutical formulation of bis-benzyl lipoateaccording to Comparative Example 1, i.e., bis-benzyl lipoate dissolvedin 1:1 Tween 80:ethanol and diluted with saline, and the other subgroupwas treated with a pharmaceutical formulation of bis-benzyl lipoateaccording to Example 1, i.e., dissolved in TEA and diluted with D5W.

TABLE 2 Revised Treatment Groups. Bis-benzyl lipoate Dose Group (mg/kg)Schedule Vehicle #mice 1 & 2 A 0.1 1x weekly TEA 4 B 1 1x weekly TEA 3 A10 1x weekly TEA 4 B 10 1x weekly Tween 80:Ethanol 3 3 A 0.1 1x weeklyTween 80:Ethanol 4 B 0.1 1x weekly TEA 3 4 A 0.1 3x weekly Tween80:Ethanol 4 B 0.1 3x weekly TEA 3 5 A 1 1x weekly Tween 80:Ethanol 3 B1 1x weekly TEA 4 6 A 1 3x weekly Tween 80:Ethanol 3 B 1 3x weekly TEA 47 A 10 1x weekly Tween 80:Ethanol 4 B 10 1x weekly TEA 3 8 A 10 3xweekly Tween 80:Ethanol 4 B 10 3x weekly TEA 3

The results showed that H-460 tumors in mice treated with 0.1-10 mg/kgof bis-benzyl lipoate in pharmaceutical formulations made according toExample 1 may be similar among each other, but may be smaller than thatin mice treated with 10 mg/kg of bis-benzyl lipoate in a pharmaceuticalformulation made according to Comparative Example 1.

Next, again the protocol was revised to change the pharmaceuticalformulation tested for anti-tumor efficacy to exclusively pharmaceuticalformulations made according to Example 1, i.e., dissolved in TEA anddiluted with D5W. There were ten treatment groups, with 8 mice in eachgroup, investigating three doses (0.1, 1 and 10 mg/kg) and three dosingschedules, as shown in Table 3 below.

TABLE 3 Second Revised Treatment Groups. Dose of bis-benzyl TreatmentGroup lipoate (mg/kg) Dose Schedule # of mice 1 0 (vehicle*) 5x weekly 82 0.1 1x weekly 8 3 0.1 3x weekly 8 4 0.1 5x weekly 8 5 1 1x weekly 8 61 3x weekly 8 7 1 5x weekly 8 8 10 1x weekly 8 9 10 3x weekly 8 10 10 5xweekly 8 *vehicle refers to D5W only

The results (as shown in FIGS. 2A-2C) showed that bis-benzyl lipoate inpharmaceutical formulations prepared according to Example 1 (TEA/D5W) at0.1, 1 and 10 mg/kg, given 1×, 3× or 5× weekly induced a similar andsignificant degree of tumor growth inhibition as compared with theresults obtained when pharmaceutical formulations prepared according toComparative Example 1 (Tween 80/ethanol/saline) were similarly tested.

EXAMPLE 2

Numerous formulations of CPI-613 were tested for their ability to killA2780 ovarian tumor cells and BXPC3 human pancreatic cancer cells usingan in vitro assay. The effect of the formulation vehicle alone on A2780ovarian tumor cells and BXPC3 human pancreatic cancer cells was alsoevaluated. The experimental procedures and results are described below.

Experimental Procedures:

1. Materials and Human Tumor Cells

Materials for the experiments were obtained through normal distributionchannels from commercial vendors. For example, Costar opaque-walledplates were obtained from Corning Costar Corporation of Cambridge, Mass.(cat. no. 3917). FLUOstar OPTIMA was obtained from BMG LABTECH ofOffenburg, Germany. CELLTITER-GLO® Luminescent Cell Viability Assaymaterials were obtained from Promega (Fisher Scientific cat no.PR-G7573). RPMI 1640 Tissue culture medium was obtained from Mediatech(Fisher Scientific cat. no. MT-10040-CV). The Fetal Bovine Serum (FBS)corresponded to Fisher Scientific cat. no. MTT35011CV, and thepenicillin and steptomycin correspond to Fisher Scientific cat. no. MT30-009-CI.

Two types of human tumor cells were used in this investigation: BXPC3human pancreatic cancer cells and A2780 human ovarian cancer cells. TheBXPC3 cells were originally obtained from American Type Cell Culture(ATCC). The ovarian tumor cells were gifts from Roswell Park CancerInstitute, Buffalo, N.Y. All tumor cells were maintained at 37° C. in ahumidified 5% CO₂ atmosphere in T75 tissue culture flasks containing 20mL of Roswell Park Memorial Institute (RPMI) 1640 containing 2 mML-glutamine, 10% fetal bovine serum (FBS) and 1% penicillin andstreptomycin (100 IU/mL penicillin and 100 μg/mL streptomycin). Thetumor cells were split at a ratio of 1:5 every 4-5 days bytrypsinization and resuspended in fresh medium in a new flask. Cellswere harvested for experiments at 70-90% confluency.

2. Preparation of CPI-613 Formulations in Different Vehicles for CellViability Assays

CPI-613 (i.e., 6,8-bis(benzylthio)octanoic acid) stock solutions atconcentrations of 8, 4, 2, 1.5 and 0.5 mM were prepared in theappropriate vehicles. The molar ratio of vehicle to CPI-613 wasmaintained at 8:1. The corresponding vehicle concentrations were 64, 32,16, 12, and 4 mM respectively. A sixty-four mM aliquot of vehicle wasdissolved in 10 mL of water. Next, 8 mM of CPI-613 was added and themixture was stirred until a clear solution was obtained. The resulting 8mM drug/64 mM vehicle formulation was then serially diluted with waterto give a 4 mM drug/32 mM vehicle and 2 mM drug/16 mM vehicle solution.The appropriate amount of water was added to the 2 mM drug/16 mM vehiclesolution to give 1.5 mM drug/12 mM vehicle and 0.5 mM drug/4 mM vehiclesolutions. A 5 μL aliquot of this stock solution was added to 95 μL oftumor cells in media. Upon dilution in the wells, the corresponding drugconcentrations were 400, 200, 100, 75 and 25 μM respectively.

3. Preparation of CPI-613 Alkali Salts and CPI-613 Ammonium Salt forCell Viability Assays

For the sodium salt and potassium salt form of CPI-613, 1 equivalent ofCPI-613 solid was dissolved in water. Sixty-two mg of CPI-613 (0.00016mol) was added to 10 mL of water, then 165 μL of 1M NaOH or KOH wasadded and stirred until a clear solution was obtained. This gave a 16 mMsolution. The resulting 16 mM solution was then serially diluted,thereby providing 8 mM and 4 mM solutions of the CPI-613 salt form. Forthe ammonium salt, vehicle to drug ratio was maintained at 8:1 and theformulation was prepared using procedures analogous to those describedabove in Section 2 relating to preparation of CPI-613 formulations indifferent vehicles.

4. Preparation of CPI-613 Formulations for in Different Vehicles forCell Viability Assays

CPI-613 formulated in 1M triethanolamine (TEA) was used as an internalcontrol during formulation screening. To prepare a 16 mM of workingstock of CPI-613, 50 mg/mL of CPI-613 (128.66 mM of CPI-613 in 1M TEA)was diluted in complete cell culture media containing 10% FBS. That is,310 μL of a 128.66 mM solution of CPI-613 in 1M TEA was added to 2.19 mLof cell culture media. This stock was further diluted serially incomplete media to give 8 mM, 4 mM 2 mM, 1.5 mM and 0.5 mM workingsolutions. On the day of testing, 5 μL of this working solution wasadded to 95 μL of cell culture media (20 fold dilution) resulting finalconcentrations of 400, 200, 100, 75 and 25 μM CPI-613, respectively. Theratio of CPI-613 to TEA was maintained at 1:8 ratio at all times.

5. Study Design

The cell viability of CPI-613 formulations formulated at a ratio of 1:8,CPI-613 to vehicle, was assessed by exposing the A2780 ovarian tumorcells and BXPC3 human pancreatic cancer cells to the following CPI-613concentrations: 400 μM (3200 μM vehicle), 200 μM (1600 μM vehicle), 100μM (800 μM vehicle), 75 μM (600 μM vehicle) and 25 μM (200 μM vehicle)The impact of corresponding vehicles at 3200, 1600 and 800 μMconcentrations was also tested in tumor cells for cell viability. Tumorcells were also treated with 25 μM, 75 μM, 100 μM, 200 μM and 400 μM ofsalt analogs of CPI-613. CPI-613 in TEA was used as an internal control.The tumor cells were treated for 24 hrs with the test article inserum-containing medium. After 24 hrs of treatment the number of viabletumor cells was determined.

6. Study Procedures

Part I: Cell Seeding for Experiments

Cells were grown to 70-90% confluency, medium was removed and the cellmonolayers were washed briefly by adding 5 mL of phosphate buffer saline(PBS) followed by aspiration. Trypsin-ethylenediaminetetraacetic acid(EDTA) (4 mL) was added to each flask, and the flask was placed in thetissue culture incubator for 5 minutes. Serum-containing medium (10 mL)was added to halt the enzymatic reactions, and cells were disaggregatedby repeated resuspension with serological pipette. The cell-containingmedium (20 μL) was added to 20 μL of 0.4% Trypan Blue solution, mixed,and 10 μL of this cell-containing mixture was placed in a chamber of thehemocytometer. The number of viable cells were determined by countingthe number of viable cells (cells that excluded Trypan blue) in the 4corner squares of the hemocytometer chamber at 100× magnification, toget the average number of cells present. The volume of cells needed wasdetermined by the following formula:

${{Volume}\mspace{14mu}{of}\mspace{14mu}{cells}\mspace{14mu}{needed}} = \frac{\#\mspace{14mu}{of}\mspace{14mu}{cells}\mspace{14mu}{need}\mspace{14mu}{for}\mspace{14mu}{the}\mspace{14mu}{assay}\mspace{11mu}({mL})}{\#\mspace{14mu}{of}\mspace{14mu}{cells}\mspace{14mu}{counted}\mspace{11mu}({mL})}$where: # of cells counted (mL)=average # of cells on hemocytometer×2(dilution factor)×10⁴.

The number of cells targeted for the study was 4×10³ per well in 100 μLof medium. The actual number of cells were counted and seeded in thewells of a 96 well-plate. The cells were incubated for ˜24 hrs beforeaddition of CPI-613.

Part II: Treatment with Test Article

For each cell line the concentrations of test articles or vehiclesspecified above was used. Five μL of the working solution for each testarticle or vehicle was added to 95 μL of cell culture media. Thecorresponding final concentrations of test articles are 20 fold morediluted than the working solutions. After exposure to the test articlesfor 24 hrs, the number of viable cells in each well was determined andthe percent of viable cells relative to control (in the absence ofCPI-613) were calculated. Additionally, a set of wells was treated withcell culture medium in the absence of cells to obtain a value forbackground luminescence. A separate set of cells was seeded at the sametime in a clear 96 well plate and observed under the microscope at 24hrs, following addition of CPI-613 to estimate the amount of cellspresent after treatment.

Part III: Determination of the Number of Viable Cells by theCELLTITER-GLO® Assay

The number of viable cells was determined by using the CELLTITER-GLO®Assay. Specifically, reagents were mixed and allowed to come to roomtemperature according to instructions from Promega, Inc. (Madison,Wis.). Cell plates were removed from the cell culture incubator and lefton the bench for 30 minutes until they reach room temperature. Aone-hundred μL aliquot CELLTITER-GLO® reagent was added to each wellwith a 12-channel Eppendorf pipettor. The cells were lysed by shakingthe plate for 2 minutes in a shaker. Then, the cells were kept at roomtemperature for 10 minutes to stabilize the luminescent signal. Theluminescence was measured using the FLUOstar OPTIMA plate reader (BMGLabtech, Inc., Durham, N.C.).

Part IV: Calculations of EC₅₀ Values

Data from luminescence readings was copied onto EXCEL spreadsheets, andcell growth relative to untreated cells was calculated, using thefollowing equation:

${\%\mspace{14mu}{growth}\mspace{14mu}{related}\mspace{14mu}{to}\mspace{14mu}{NT}} = {\frac{{mean}\mspace{14mu}{luminescence}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{test}\mspace{14mu}{article}}{{mean}\mspace{14mu}{luminescence}\mspace{14mu}{untreated}} \times 100\%}$

The calculated values were imported into SigmaPlot, v11. AFour-Parameter Logistic Curve of the “mean relative cell growth as afunction of the concentrations of the test articles” was generated. TheEC₅₀ values were determined from the curves. The R-squared valueprovides an indication of the degree of fitness of data to the curve.For each experiment, the percentage of viable cells was be expressed asthe mean of 9 replicates. The average was calculated by taking themean±standard deviation of the three experiments.

Results:

Results from the above-described experiment are presented below insections according to the nature of the experiment.

Part I: Impact of Formulation Vehicles on Tumor Cell Viability

The vehicles used in CPI-613 formulations were assessed for their impacton tumor cell viability at a concentration of 3200, 1600 and 800 μM. Theresults from these assays are shown in Tables 4 and 5 below. The dataindicate that at vehicle concentrations of 800 μM and 1600 μM all thevehicles tested did not have a significant effect on tumor cell kill inboth A2780 & BXPC3 cell lines. At a vehicle concentration of 3200 μM,the following vehicles appear to have come cell killing effects:triisopropanolamine, ethylenediamine, and diisopropanolamine. Thevehicles diethanolamine, meglumine, 3-amino-1-proponol, triethylamine,aminopropanediol, tris base, lysine, diethylamine and piperazine did notshow a significant impact on cancer cell killing at a concentration of3200 μM, and it is contemplated that these vehicles can be used toformulate CPI-613 even at very high concentrations of the vehicle.

In particular, Table 4 shows the impact of formulation vehicles on A2780tumor cell viability. Table 5 shows the impact of formulation vehicleson BXPC3 tumor cell viability. The results are the average of threeexperiments with each experiment having 4 replicates. The symbol +indicates a tumor cell viability of >95%, the symbol ++ indicates atumor cell viability of >85% up to 95%, the symbol +++ indicates a tumorcell viability of >70% up to 85%, the symbol ++++ indicates a tumor cellviability of ≤70%. Tumor cell viability refers to the percentage oftumor cells in the population that remain alive following treatment withthe test substance. For example, a tumor cell viability of >95% meansthat greater than 95% of the cells in the population are alive followingtreatment with the test substance. A tumor cell viability of <70%percent means that less than 70% of the cells in the population arealive following treatment with the test substance.

TABLE 4 A2780 Tumor Cell Viability in the Presence of VariousFormulation Vehicles. Vehicle A2780 Cell Viability Concentration 800 μM1600 μM 3200 μM Non Treated + + + Ethanolamine + + + Diethanolamine ++++ + Meglumine + + + 3-amino-1-proponol + + ++ Triisopropanolamine + ++++ Triethylamine + + + Aminopropanediol + + + Tris base + + +Ethylenediamine ++ ++ ++++ Lysine + + + Diisopropanolamine + + ++++Diethylamine + + ++ Piperazine ++ +++ ++

TABLE 5 BXPC3 Tumor Cell Viability in the Presence of VariousFormulation Vehicles. Vehicle BXPC3 Cell Viability Concentration 800 μM1600 μM 3200 μM Non Treated + + + Ethanolamine ++ ++ ++ Diethanolamine++ ++ ++ Meglumine ++ ++ ++ 3-amino-1-proponol + ++ ++Triisopropanolamine + + +++ Triethylamine + + + Aminopropanediol + + +Tris base + + + Ethylenediamine + +++ ++++ Lysine + + +Diisopropanolamine + + +++ Diethylamine + +++ ++++ Piperazine ++ ++ +++Part II: Impact of CPI-613 Formulations on Cell Viability

The effect of different CPI-613 formulations prepared using clinicallyapproved vehicles was tested. The ratio of CPI-613 to vehicle wasmaintained at a 1:8 ratio. The cell viability of tumor cells, that wereexposed to different CPI-613 concentrations ranging from 25-400 μM weremeasured using CELLTITER-GLO® Assay. The results of this experiment aresummarized in Tables 6-7 and FIGS. 3-11. In particular, the data inTables 6-7 are the average of 2-3 experiments each having 4 replicateseach. The percent cell viability was determined compared to non-treatedcontrol, and the ratio of CPI-613 to vehicle was maintained at 1:8 inthese experiments. It is noted, however, that although a 1:8 ratio ofCPI-613 to vehicle was used in these formulations, it is contemplatedthat formulations having a lower ratio of CPI-613 to vehicle may beamenable in certain instances.

In Tables 6-7, the symbol + indicates a tumor cell viability of >95%,the symbol ++ indicates a tumor cell viability of >70% up to 95%, thesymbol +++ indicates a tumor cell viability of >30% up to 70%, thesymbol ++++ indicates a tumor cell viability of >10% up to 30%, and thesymbol +++++ indicates a tumor cell viability of ≤10%.

TABLE 6 Impact of CPI-613 Formulations on A2780 Cell Viability. A2780Cell Viability at Certain Concentrations of CPI-613 & Vehicle CPI-613Concentration (μM) 0 25 75 100 200 400 Vehicle Concentration (μM) 800200 600 800 1600 3200 Not Treated + + + + + + CPI-613 in + + + ++ +++++++++ triethanolamine CPI-613 in + + ++ ++ +++ +++++ ethanolamineCPI-613 in + + ++ ++ ++++ +++++ diethanolamine CPI-613 in + + ++ ++ +++++++++ meglumine CPI-613 in 3-amino- + + ++ ++ ++++ +++++ 1-proponolCPI-613 in + ++ ++ ++ +++ +++++ triethylamine CPI-613 in ++ + ++ +++++++ +++++ ethylenediamine CPI-613 in lysine + + ++ ++ +++ +++++ CPI-613in + + ++ ++ ++++ +++++ diisopropanolamine CPI-613 in + ++ ++ ++ ++++++++ diethylamine

TABLE 7 Impact of CPI-613 Formulations on BXPC3 Cell Viability. BXPC3Cell Viability at Certain Concentrations of CPI-613 & Vehicle CPI-613Concentration (μM) 0 25 75 100 200 400 Vehicle Concentration (μM) 800200 600 800 1600 3200 Not Treated + + + + + + CPI-613 in + + ++ ++ +++++++++ triethanolamine CPI-613 in + + + ++ +++ +++++ ethanolamine CPI-613in + + + ++ +++ +++++ diethanolamine CPI-613 in + + ++ ++ +++ +++++meglumine CPI-613 in 3-amino- + + ++ ++ +++ +++++ 1-proponol CPI-613in + + ++ ++ +++ +++++ triethylamine CPI-613 in ++ + ++ ++ +++ +++++ethylenediamine CPI-613 in lysine + + + ++ +++ +++++ CPI-613 in + + + +++++ +++++ diisopropanolamine CPI-613 in + + ++ ++ +++ +++++ diethylamine

Results from the experiment indicate, for example, that exposure of thetest cancer cells to the CPI-613 in triethanolamine formulation at aconcentration of 100 μM resulted in the death of approximately 20% ofthe cancer cells in the population. Exposure of the test cancer cells tothe CPI-613 in triethanolamine formulation at a concentration of 200 μMresulted in the death of approximately 70-80% of the cancer cells in thepopulation, and exposure of the test cancer cells to the CPI-613 intriethanolamine formulation at a concentration of 400 μM resulted in thedeath of approximately 100% of the cancer cells in the population.CPI-613 in any one of ethanolamine, diethanolamine, meglumine,3-amino-1-proponol, triethylamine, ethylenediamine, lysine,diisopropanolamine and diethylamine at a concentration of 200 μMconcentration resulted in the death of more than 50% of the cancercells, and, at a concentration of 400 μM all of the formulations causeddeath of 100% of the cancer cells based on detection methods.

Part III: Impact of CPI-613 Alkali Salts and CPI-613 Ammonium SaltFormulations on Cell Viability

The effect of different CPI-613 salt formulations described above weretested. The viability of tumor cells exposed to CPI-613 saltformulations was measured using a CELLTITER-GLO® Assay. Experiments wereconducted measuring cell viability when the tumor cells were exposed toCPI-613 salt formulations ranging in concentration from 25-400 μM.Moreover, the ratio of CPI-613 to base was maintained at 1:1 in thesodium salt and the potassium salt formulation, whereas in the ammoniumsalt formulation the ratio was 1:8. The results are shown in Tables 8-11and FIGS. 12-14 and are an average of three experiments each having fourreplicates. The percent cell viability was determined compared to acontrol experiment where the cells were not treated with CPI-613 saltformulation. In Tables 8-11, the symbol + indicates a tumor cellviability of >95%, the symbol ++ indicates a tumor cell viabilityof >70% up to 95%, the symbol +++ indicates a tumor cell viabilityof >30% up to 70%, the symbol ++++ indicates a tumor cell viabilityof >10% up to 30%, and the symbol +++++ indicates a tumor cell viabilityof <10%.

TABLE 8 Impact of CPI-613 Alkali Salt Formulations on Viability of A2780Cells. A2780 Cell Viability at Certain Concentrations CPI-613Concentration (μM) 0 25 75 100 200 400 Not treated + + + + + + CPI-613sodium salt ++ ++ ++ ++ ++++ +++++ CPI-613 potassium ++ ++ ++ ++ ++++++++ salt

TABLE 9 Impact of CPI-613 Alkali Salt Formulations on Viability of BXPC3Cells. BXPC3 Cell Viability at Certain Concentrations CPI-613Concentration (μM) 0 25 75 100 200 400 Not treated + + + + + + CPI-613sodium salt + + + ++ +++ +++++ CPI-613 potassium + + ++ ++ +++ +++++salt

TABLE 10 Impact of CPI-613 Ammonium Salt Formulations on Viability ofA2780 Cells. A2780 Cell Viability at Certain Concentrations CPI-613Concentration (μM) 0 25 75 100 200 400 Not treated + + + + + + CPI-613in + + + ++ ++++ +++++ triethanolamine (control) CPI-613 ammonium + + +++ ++++ +++++ salt

TABLE 11 Impact of CPI-613 Ammonium Salt Formulations on Viability ofBXPC3 Cells. BXPC3 Cell Viability at Certain Concentrations CPI-613Concentration (μM) 0 25 75 100 200 400 Not treated + + + + + + CPI-613in + + ++ ++ ++++ +++++ triethanolamine (control) CPI-613ammonium + + + + +++ +++++ salt

Results from the experiment indicate, for example, that the CPI-613sodium salt and CPI-613 potassium salt formulation provided almost 65%cell kill (i.e., death of almost 65% of the cancer cells in thepopulation) in A2780 cells and almost 50% cell kill in BXPC3 cells whenthe cells where exposed to these CPI-613 salt formulations at 200 μM.Further, when the A2780 cells and BXPC3 cells were exposed to theseCPI-613 salt formulations at a concentration of 400 μM, all the cellswere killed for both cell lines. The CPI-613 ammonium salt formulationprovided approximately 70-80% cell kill when the cells were exposed tothe formulation at concentration 200 μM, and 100% cell kill was observedfollowing administration of the CPI-613 ammonium salt formulation at aconcentration of 400 μM.

EXAMPLE 3

In this example, the type of cell death that occurs followingadministration of CPI-613 triethanolamine and CPI-613 sodium salt wasevaluated. The experimental procedures and results are described below.

Experimental Procedures:

Part I—Equipment

Subsequent to propidium iodide (PI)/Annexin Alexa Fluor 488 staining,necrotic and apoptotic BXPC3 cells were assessed with the aid ofFluorescence Activated Cell Sorting (FACS) Scan Flow Cytometer (FACSCalibur Instrument, BD Biosciences, San Jose, Calif.) and CellQuestsoftware (BD Biosciences, San Jose, Calif.). This software identifiesthe quantity of DNA content. Apoptotic cells will stain Annexin Vpositive but PI negative because the PI cannot cross the intact cellmembrane. Necrotic cells will stain positive for both Annexin V and PIbecause the cell membrane is not intact.

Part II—Materials

The reagent used for PI/Annexin Alexa Fluor 488 Staining of BXPC3 Cellswas supplied by the Invitrogen/Molecular Probes Vybrant Apoptosis AssayKit #2 cat # V13241.

Part III—Human Cancer Cells:

BxPC3 human pancreatic cancer cells were used in this investigation.These tumor cells were originally obtained from American Type CellCulture (ATCC, Manassas, Va.). These cells had been tested negative forviral contamination using the Mouse Antibody Production (MAP) test,performed by Charles River Labs Molecular Division, upon the receipt ofthe tumor cells from ATCC. The tumor cells were maintained at 37° C. ina humidified 5% CO₂ atmosphere in T225 tissue culture flasks containing50 mL of Roswell Park Memorial Institute (RPMI)-1640 solution with 10%Fetal Bovine Serum (FBS) and 2 mM L-glutamine. Cells were split at aratio of 1:10 every 2-3 days by trypsinization and resuspended in freshmedium in a new flask. Cells were harvested for experiments in the sameway at 70-90% confluency.

Part IV—Plating of BxPC3 Cells

BxPC3 cells were plated at a density of 0.3×10⁶ cells per well in asterile 6-well tissue culture plate (Falcon multiwell plate cat #353046)48 hours prior to drug treatment.

Part IV—Treatment Groups

The following treatment groups were utilized in this experiment: 1)Non-treated BxPC3 cells not drug (control), 2) Triethanolamine (TEA)with no drug as a stock solution of 700 mM with final concentration onthe cells being 2.1 mM, 3) CPI-613 in TEA as a stock solution of 100 mMof CPI-613 in 700 mM TEA with final concentration on the cells being 300μM of CPI-613 in 2.1 mM TEA, 4) Sodium salt of CPI-613 as a stocksolution of 100 mM with final concentration on the cells being 300 μM,and 5) 0.9% NaCl with no CPI-613.

Part V—Design of Experiment:

The experiment was conducted in accordance with the steps identifiedbelow:

1) BxPC3 cells were plated at a density of 0.3×10⁶ cells per well in asterile 6-well tissue culture plate (Falcon multiwell plate cat #353046)48 hours prior to drug treatment.

2) A 1 mL aliquot of each test condition was added to correspondingwell. Each treatment condition was run in duplicate. Cells wereincubated for 24 hours with test conditions prior to PI/AnnexinStaining.

3) After 24 hours, cells, including floating cells, with media wereharvested and centrifuged at 1,400 RPM for 5 minutes.

4) For all samples the supernatant was aspired off, and the pellet wasresuspended in 3 mL PBS and then centrifuged again at 1,400 RPM for 5minutes, for a total of 2 washes.

5) After the last wash, each pellet was then resuspended with 200 μL 1×Annexin V binding buffer, 1 μL PI, and 3 μL Annexin Alexa Fluor 488.Cells were incubated with PI/Annexin V for 8 minutes.

6) The reaction was stopped by adding 400 μL 1× Annexin V bindingbuffer.

7) Samples were then taken to FACS facility to be read on the FACS ScanFlow Cytometer.

Results:

Data from the experiment is shown in FIGS. 15-17. The data seems toindicate a difference between the type of cell death (early apoptosis,late apoptosis, or necrosis) observed when comparing the TEA formulationof CPI-613 to the sodium salt formulation of CPI-613. The results fromthe TEA formulation indicate an increase in the number of cells in thestate of necrotic death versus the number of cells in the state ofapoptotic death. In contrast, the sodium salt formulation of CPI-613showed a higher number of cells in the state of apoptotic death versusthe number of cells in the state of necrotic death.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientificarticles referred to herein is incorporated by reference for allpurposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting the invention described herein. The scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

What is claimed is:
 1. A method of treating a cancer selected from thegroup consisting of a carcinoma, sarcoma, myeloma, lymphoma, leukemia, amixed type thereof, and a cancer of the pancreas in a human patient inneed thereof, comprising the step of intravenously administering to thepatient a pharmaceutical composition comprising a therapeuticallyeffective amount of 6,8-bis(benzylthio)octanoic acid or an ion pairthereof, and a pharmaceutically acceptablediluent for solubilizing6,8-bis(benzylthio)octanoic acid or an ion pair thereof.
 2. The methodof claim 1, wherein the cancer is a cancer of the pancreas.
 3. Themethod of claim 1, wherein the cancer is a lymphoma.
 4. The method ofclaim 1, wherein the cancer is a leukemia.
 5. The method of claim 1,wherein the cancer is a carcinoma.
 6. The method of claim 1, wherein thecancer is a sarcoma.
 7. The method of claim 1, wherein the cancer is amyeloma.
 8. The method of claim 1, wherein the6,8-bis(benzylthio)octanoic acid or an ion pair thereof is administeredat a dose of 20 mg/m² to 2500 mg/m².
 9. The method of claim 1, whereinthe 6,8-bis(benzylthio)octanoic acid or an ion pair thereof isadministered at a dose of 0.25 mg/m² to 3 g/m².
 10. The method of claim2, wherein the 6,8-bis(benzylthio)octanoic acid or an ion pair thereofis administered at a dose of 20 mg/m² to 500 mg/m².
 11. The method ofclaim 2, wherein the 6,8-bis(benzylthio)octanoic acid or an ion pairthereof is administered at a dose of 500 mg/m² to 700 mg/m².
 12. Themethod of claim 2, wherein the 6,8-bis(benzylthio)octanoic acid or anion pair thereof is administered at a dose of 500 mg/m².
 13. The methodof claim 3, wherein the 6,8-bis(benzylthio)octanoic acid or an ion pairthereof is administered at a dose of 20 mg/m² to 2500 mg/m².
 14. Themethod of claim 3, wherein the 6,8-bis(benzylthio)octanoic acid or anion pair thereof is administered at a dose of 0.25 mg/m² to 3 g/m². 15.The method of claim 3, wherein the 6,8-bis(benzylthio)octanoic acid oran ion pair thereof is administered at a dose of 2500 mg/m².
 16. Themethod of claim 4, wherein the 6,8-bis(benzylthio)octanoic acid or anion pair thereof is administered at a dose of 20 mg/m² to 2500 mg/m².17. The method of claim 4, wherein the 6,8-bis(benzylthio)octanoic acidor an ion pair thereof is administered at a dose of 0.25 mg/m² to 3g/m².
 18. A method of treating carcinoma, sarcoma, myeloma, lymphoma,leukemia, or a mixed type thereof, or a cancer of the pancreas in ahuman patient in need thereof, comprising the step of intravenouslyadministering to the patient a pharmaceutical composition comprising atherapeutically effective amount of 6,8-bis(benzylthio)octanoic acid oran ion pair thereof, and a pharmaceutically acceptable aqueous diluentfor solubilizing 6,8-bis(benzylthio)octanoic acid or an ion pairthereof.
 19. The method of claim 18, wherein the6,8-bis(benzylthio)octanoic acid or an ion pair thereof is administeredfor the treatment of a cancer of the pancreas.
 20. The method of claim18, wherein the 6,8-bis(benzylthio)octanoic acid or an ion pair thereofis administered for the treatment of lymphoma.
 21. The method of claim18, wherein the 6,8-bis(benzylthio)octanoic acid or an ion pair thereofis administered for the treatment of leukemia.
 22. The method of claim18, wherein the 6,8-bis(benzylthio)octanoic acid or an ion pair thereofis administered for the treatment of carcinoma.
 23. The method of claim18, wherein the 6,8-bis(benzylthio)octanoic acid or an ion pair thereofis administered for the treatment of sarcoma.
 24. The method of claim18, wherein the 6,8-bis(benzylthio)octanoic acid or an ion pair thereofis administered for the treatment of myeloma.
 25. The method of claim18, wherein the 6,8-bis(benzylthio)octanoic acid or an ion pair thereofis administered at a dose of 20 mg/m² to 2500 mg/m².
 26. The method ofclaim 18, wherein the 6,8-bis(benzylthio)octanoic acid or an ion pairthereof is administered at a dose of 0.25 mg/m² to 3 g/m².
 27. Themethod of claim 19, wherein the 6,8-bis(benzylthio)octanoic acid or anion pair thereof is administered at a dose of 20 mg/m² to 500 mg/m². 28.The method of claim 19, wherein the 6,8-bis(benzylthio)octanoic acid oran ion pair thereof is administered at a dose of 500 mg/m² to 700 mg/m².29. The method of claim 19, wherein the 6,8-bis(benzylthio)octanoic acidor an ion pair thereof is administered at a dose of 500 mg/m².
 30. Themethod of claim 20, wherein the 6,8-bis(benzylthio)octanoic acid or anion pair thereof is administered at a dose of 20 mg/m² to 2500 mg/m².31. The method of claim 20, wherein the 6,8-bis(benzylthio)octanoic acidor an ion pair thereof is administered at a dose of 0.25 mg/m² to 3g/m².
 32. The method of claim 20, wherein the6,8-bis(benzylthio)octanoic acid or an ion pair thereof is administeredat a dose of 2500 mg/m².
 33. The method of claim 21, wherein the6,8-bis(benzylthio)octanoic acid or an ion pair thereof is administeredat a dose of 20 mg/m² to 2500 mg/m².
 34. The method of claim 21, whereinthe 6,8-bis(benzylthio)octanoic acid or an ion pair thereof isadministered at a dose of 0.25 mg/m² to 3 g/m².
 35. The method of claim25, wherein the pharmaceutically acceptable aqueous diluent is adextrose solution which contains an amount of dextrose ranging from 2.5%to 10% by weight.
 36. The method of claim 26, wherein thepharmaceutically acceptable aqueous diluent is a dextrose solution whichcontains an amount of dextrose ranging from 2.5% to 10% by weight. 37.The method of claim 27, wherein the pharmaceutically acceptable aqueousdiluent is a dextrose solution which contains an amount of dextroseranging from 2.5% to 10% by weight.
 38. The method of claim 28, whereinthe pharmaceutically acceptable aqueous diluent is a dextrose solutionwhich contains an amount of dextrose ranging from 2.5% to 10% by weight.39. The method of claim 29, wherein the pharmaceutically acceptableaqueous diluent is a dextrose solution which contains an amount ofdextrose ranging from 2.5% to 10% by weight.
 40. The method of claim 30,wherein the pharmaceutically acceptable aqueous diluent is a dextrosesolution which contains an amount of dextrose ranging from 2.5% to 10%by weight.
 41. The method of claim 31, wherein the pharmaceuticallyacceptable aqueous diluent is a dextrose solution which contains anamount of dextrose ranging from 2.5% to 10% by weight.
 42. The method ofclaim 32, wherein the pharmaceutically acceptable aqueous diluent is adextrose solution which contains an amount of dextrose ranging from 2.5%to 10% by weight.
 43. The method of claim 33, wherein thepharmaceutically acceptable aqueous diluent is a dextrose solution whichcontains an amount of dextrose ranging from 2.5% to 10% by weight. 44.The method of claim 34, wherein the pharmaceutically acceptable aqueousdiluent is a dextrose solution which contains an amount of dextroseranging from 2.5% to 10% by weight.